Matrix-assisted spatial analysis of biological samples

Abstract

The present disclosure relates in some aspects to methods and compositions for matrix-assisted spatial analysis, where a biological sample is embedded in a matrix (e.g., a hydrogel matrix) and a target nucleic acid from the biological sample is tethered to the matrix, followed by removing the biological sample (e.g., by clearing the sample) from the matrix while the target nucleic acid remains tethered to the matrix. After sample removal, one or more probes can be hybrized to the tethered target nucleic acid. The target nucleic acid, the one or more probes, a product of the target nucleic acid, and / or a product of the one or more probes can be released from the matrix for capturing on an array, spatial barcoding on the array, and subsequent sequence analysis of spatially barcoded molecules, for example, using a next-generation sequencing method.

Description

202412020440 MATRIX-ASSISTED SPATIAL ANALYSIS OF BIOLOGICAL SAMPLES CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 597,578, filed on November 9, 2023, entitled “MATRIX-ASSISTED SPATIAL ANALYSIS OF BIOLOGICAL SAMPLES,” which is herein incorporated by reference in its entirety for all purposes. FIELD

[0002] The present disclosure relates in some aspects to methods and compositions for spatial analysis of a target molecule in a biological sample (e.g., a cell or tissue sample), involving tethering a nucleic acid molecule (e.g., a cellular DNA or RNA molecule, a nucleic acid probe, or an analyte capture agent comprising i) an analyte binding moiety and ii) an analyte associated oligonucleotide which comprises an analyte capture sequence and an analyte binding moiety barcode) to a matrix, removing the biological sample (e.g., by clearing at least a subset of proteins and / or lipids from the matrix), and capturing the nucleic acid molecule or a proxy thereof on an array for analysis, for instance, in order to determine a spatial profile of the target molecule in the biological sample. BACKGROUND

[0003] Cells within a tissue of a subject have differences in cell morphology and / or function due to varied analyte levels (e.g., gene and / or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell’s position relative to neighboring cells or the cell’s position relative to the tissue microenvironment) can affect, e.g., the cell’s morphology, differentiation, fate, viability, proliferation, behavior, signaling and cross- talk with other cells in the tissue.

[0004] Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provide substantial analyte data for dissociated tissue (e.g., single cells) but fail to provide information regarding the positions of the single cells in a parent biological sample (e.g., tissue sample). Improved methods for spatial analysis of analytes in a biological sample are needed. Provided herein are methods and compositions that address such and other needs. 1ny-2785575202412020440 BRIEF SUMMARY

[0005] Process steps in spatial transcriptomic profiling of tissue samples can lead to loss of target molecules (e.g., RNA transcripts, nucleic acid probes targeting the transcripts, etc.). Such steps can include decrosslinking of fixed tissue samples, and probe hybridization to target nucleic acids such as RNA transcripts. Extended exposure to high temperatures (e.g., in sample decrosslinking and / or probe hybridization) can cause excess diffusion and transcript mislocalization of target molecules throughout, and even outside of, the tissue samples being profiled. Mislocalization of target molecules can lead to reduced sensitivity and decreased resolution of the spatial profiling of the tissue samples, which can lead to compromised accuracy in determining the presence, absence, or amount of the target molecule at locations in the sample. Thus, improved methods for spatial analysis of analytes in a biological sample are needed.

[0006] In some embodiments, provided herein are matrix-assisted methods involving tethering nucleic acid molecules in a biological sample, such as RNA molecules, to a matrix such as a hydrogel embedding the biological sample. In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: tethering a target nucleic acid in the biological sample to a matrix embedding the biological sample; removing a subset of cellular components from the biological sample in the matrix; releasing the target nucleic acid from the matrix; hybridizing or ligating the released target nucleic acid to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0007] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a matrix; b) tethering a target nucleic acid in the biological sample to the matrix; c) removing a subset of cellular components from the biological sample in the matrix; d) releasing the target nucleic acid from the matrix; e) hybridizing or ligating the released target nucleic acid to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and f) determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0008] In any of the preceding embodiments, the target nucleic acid can comprise DNA and / or RNA. In any of the preceding embodiments, the target nucleic acid can be mRNA.ny-2785575202412020440

[0009] In any of the preceding embodiments, the matrix can comprise a hydrogel matrix. In any of the preceding embodiments, the tethering can comprise tethering a 3’ end of the target nucleic acid to the matrix. In any of the preceding embodiments, the tethering can comprise tethering a 5’ end of the target nucleic acid to the matrix. In any of the preceding embodiments, the tethering can comprise enzymatic tethering. In any of the preceding embodiments, the tethering can comprise non-enzymatic tethering. In any of the preceding embodiments, the tethering can comprise a periodate oxidation reaction or a reaction with boronic acid. In any of the preceding embodiments, the tethering can comprise tethering the target nucleic acid to the matrix via a linker. In any of the preceding embodiments, the linker can comprise a cleavable linker. In any of the preceding embodiments, the linker can comprise a disulfide bond. In any of the preceding embodiments, the matrix can be formed using N,N′- Bis(acryloyl)cystamine (BAC) as a crosslinker.

[0010] In any of the preceding embodiments, the removing of the subset of cellular components from the biological sample can comprise removing at least a subset of ribosomes from the biological sample.

[0011] In any of the preceding embodiments, the releasing can comprise contacting the matrix with a chemical agent configured to cleave a cleavable linker between the target nucleic acid and the matrix.

[0012] In any of the preceding embodiments, the releasing can comprise illuminating the matrix with light, thereby cleaving a cleavable linker between the target nucleic acid and the matrix.

[0013] In any of the preceding embodiments, the releasing can comprise altering the pH of a solution in contact with the matrix, thereby releasing the tethered target nucleic acid.

[0014] In any of the preceding embodiments, the target nucleic acid can be tethered to the matrix via a boronate ester bond. In any of the preceding embodiments, the boronate ester bond can be formed between a boronic acid moiety and 3’ diols of the target nucleic acid, wherein the target nucleic acid is RNA. In any of the preceding embodiments, the matrix can comprise a boronic acid-based hydrogel matrix.

[0015] In any of the preceding embodiments, the matrix can comprise a temperature- responsive polymer. In any of the preceding embodiments, the matrix can comprise a pH- responsive polymer. In any of the preceding embodiments, the matrix can comprise any-2785575202412020440 temperature- and pH-responsive polymer. In any of the preceding embodiments, the matrix can comprise a poly(N-isopropylacrylamide). In any of the preceding embodiments, the matrix can comprise a poly(N-isopropylacrylamide) and / or a poly(N-vinylcaprolactam). In any of the preceding embodiments, the matrix can comprise a temperature- and / or pH-responsive polymer based on a poly(N-isopropylacrylamide) and / or a poly(N-vinylcaprolactam). In any of the preceding embodiments, the releasing can comprise exposing the matrix to a temperature of about 40°C to about 75°C, wherein the matrix is a hydrogel matrix comprising poly(N- isopropylacrylamide), thereby releasing the tethered target nucleic acid.

[0016] In any of the preceding embodiments, the target nucleic acid can comprise a cellular nucleic acid in the biological sample, a product of the cellular nucleic acid generated in the biological sample, a nucleic acid probe that directly or indirectly binds to the cellular nucleic acid, or a product of the nucleic acid probe generated in the biological sample.

[0017] In any of the preceding embodiments, the matrix and the biological sample embedded therein can be on a first solid support, and the spatial array can be on a second solid support.

[0018] In any of the preceding embodiments, the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, can be determined using nucleic acid sequencing. In any of the preceding embodiments, the nucleic acid sequencing can comprise sequencing by synthesis, sequencing by ligation, sequencing by binding, sequencing by avidity, sequencing by hybridization, or any combination thereof.

[0019] In any of the preceding embodiments, the method can further comprise directing the released target nucleic acid to the capture probe. In any of the preceding embodiments, the method can comprise directing the released target nucleic acid to the capture probe via a capacitor, thereby generating an electrophoretic migration of the released target nucleic acid from the matrix to the spatial array. In any of the preceding embodiments, at least one plate of the capacitor can comprise a coating of indium tin oxide.

[0020] In any of the preceding embodiments, the matrix can be a first hydrogel matrix and the capture probe can be affixed on the spatial array and embedded in a second hydrogel matrix. In any of the preceding embodiments, the first hydrogel matrix and the secondny-2785575202412020440 hydrogel matrix can be of the same composition. In any of the preceding embodiments, the first hydrogel matrix and the second hydrogel matrix can be of different compositions.

[0021] In any of the preceding embodiments, the method can comprise determining a spatial gene expression profile of the target nucleic acid in the biological sample. In any of the preceding embodiments, a plurality of different target nucleic acids can be tethered to the matrix in a), released from the matrix in c), and hybridized or ligated to capture domains of capture probes on the spatial array.

[0022] In any of the preceding embodiments, the method can comprise digesting and / or clearing the embedded biological sample from the matrix. In any of the preceding embodiments, the biological sample can be a fresh tissue sample, a frozen tissue sample, or a fixed tissue sample. In any of the preceding embodiments, the biological sample can be a fresh frozen tissue section or a formalin-fixed paraffin-embedded tissue section. In any of the preceding embodiments, the method can comprise fixing the biological sample prior to embedding in the matrix. In any of the preceding embodiments, the method can comprise decrosslinking the embedded biological sample prior to or during the removing of the subset of cellular components.

[0023] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: a) contacting a biological sample comprising a target nucleic acid with an attachment agent comprising a boronic acid moiety capable of covalently reacting with at least one 2’,3’ vicinal diol of the target nucleic acid and an attachment moiety capable of attaching covalently or noncovalently to a matrix-forming agent in the biological sample, wherein the biological sample and the attachment agent are contacted under conditions suitable to form a covalent bond between the boronic acid moiety and the 2’,3’ vicinal diol of the target nucleic acid; b) contacting the biological sample with the matrix-forming agent, thereby forming a matrix embedding the biological sample and tethering the target nucleic acid to the matrix; c) clearing the embedded biological sample from the matrix; d) releasing the target nucleic acid from the matrix; e) capturing the target nucleic acid by a capture probe on a spatial array, wherein the capture probe comprises a capture domain and a spatial barcode; and f) determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof.ny-2785575202412020440

[0024] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: a) contacting the biological sample comprising a fragmented target nucleic acid with a 3’ phosphatase to provide a fragmented target nucleic acid comprising a 2’,3’-vicinal diol moiety; b) contacting the biological sample with a formylation reagent, wherein the formylation reagent converts the 2’,3’-vicinal diol moiety into a 2’,3’-dialdehyde moiety; c) contacting the biological sample with an attachment agent and a matrix-forming agent, the attachment agent comprising at least one aldehyde-reactive group capable of forming a covalent bond with at least one aldehyde of the 2’,3’-dialdehyde moiety of the fragmented target nucleic acid and an attachment moiety capable of attaching covalently or noncovalently to the matrix-forming agent, thereby forming a matrix embedding the biological sample and tethering the fragmented target nucleic acid to the matrix; d) clearing the embedded biological sample from the matrix; e) releasing the fragmented target nucleic acid from the matrix; f) hybridizing the fragmented target nucleic acid to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and g) determining the sequence of all or a portion of the fragmented target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In any of the preceding embodiments, the formylation reagent can comprise sodium (meta)periodate.

[0025] In any of the preceding embodiments, the capture probe can further comprise a cleavage domain, a functional domain, a unique molecular identifier, or a combination thereof.

[0026] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) applying a first probe and a second probe to the hydrogel matrix, wherein the first probe comprises a functional sequence and a sequence substantially complementary to a first portion of the target RNA and the second probe comprises a sequence substantially complementary to a second portion of the target RNA and a capture sequence that is configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; d) hybridizing the first probe and the second probe to the target RNA tethered to the hydrogel matrix; e) ligating together the first probe and the second probe hybridized to the target RNA tethered to the hydrogel matrix, thereby generating a ligation product that is a proxy of the targetny-2785575202412020440 RNA; f) releasing the ligation product from the hydrogel matrix and hybridizing or ligating the released ligation product to the capture domain of the capture probe on the spatial array; and g) determining a spatial transcriptomic profile for the biological sample by determining a sequence of all or a portion of the ligation product, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof.

[0027] In any of the preceding embodiments, the biological sample can be a crosslinked biological sample prior to embedding the biological sample in the hydrogel matrix. In any of the preceding embodiments, the crosslinked biological sample can be embedded in the hydrogel matrix and the target RNA in the crosslinked biological sample can be tethered to the hydrogel matrix, prior to removing the embedded biological sample from the hydrogel matrix (e.g., by removing a subset of cellular components from the biological sample in the matrix). In any of the preceding embodiments, between the embedding and the removing, the method can comprise decrosslinking the crosslinked biological sample embedded in the hydrogel matrix. In any of the preceding embodiments, prior to a), the biological sample can be a fixed tissue sample. In any of the preceding embodiments, prior to a), the biological sample can be a formalin-fixed paraffin-embedded (FFPE) tissue section.

[0028] In any of the preceding embodiments, the target RNA can be 3’ tethered to the hydrogel matrix. In any of the preceding embodiments, the target RNA can be 5’ tethered to the hydrogel matrix.

[0029] In any of the preceding embodiments, the functional sequence in the first probe can be in a 5’ overhang upon hybridization of the first probe to the target RNA. In any of the preceding embodiments, the functional sequence in the first probe can comprise a primer binding sequence or a complement thereof. In any of the preceding embodiments, the capture sequence in the second probe can be in a 3’ overhang upon hybridization of the second probe to the target RNA. In any of the preceding embodiments, the capture sequence in the second probe can comprise a 3’ polyadenine sequence.

[0030] In any of the preceding embodiments, the first probe and the second probe can be ligated using the target RNA as a template with or without gap filling prior to the ligation. In any of the preceding embodiments, the first probe and the second probe can be ligated using the target RNA as a template with gap filling prior to the ligation, wherein the gap filling comprisesny-2785575202412020440 hybridization of a gap-fill oligonucleotide to the target RNA and / or primer extension of the first probe or the second probe by a polymerase.

[0031] In any of the preceding embodiments, the releasing can comprise releasing the tethered RNA from the hydrogel matrix. In any of the preceding embodiments, the releasing can comprise releasing the ligation product from the target RNA, with or without cleaving or degrading the target RNA, and with or without releasing the target RNA from the hydrogel matrix. In any of the preceding embodiments, the releasing can comprise contacting the hydrogel matrix with an RNase H.

[0032] In any of the preceding embodiments, the capture domain in the capture probe can comprise a 3’ poly(dT) sequence.

[0033] In any of the preceding embodiments, the method can comprise generating a spatially labeled polynucleotide comprising (i) from 5’ to 3’, a sequence of the released ligation product and a complementary sequence of the spatial barcode, or (ii) from 5’ to 3’, a sequence of the spatial barcode and a complementary sequence of the released ligation product.

[0034] In any of the preceding embodiments, the spatially labeled polynucleotide or a portion thereof can be released from the spatial array for analysis. In any of the preceding embodiments, the spatially labeled polynucleotide or a portion thereof released from the spatial array can be analyzed by nucleic acid sequencing.

[0035] In any of the preceding embodiments, the method can comprise imaging the spatial array using fluorescence microscopy. In any of the preceding embodiments, the method can comprise amplifying the released ligation product or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released ligation product or the spatially labeled polynucleotide or an amplification product thereof. In any of the preceding embodiments, the method can comprise performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array.

[0036] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) releasing the target RNA from the hydrogel matrix; d) hybridizing or ligating the released target RNA to a capture domain of any-2785575202412020440 capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and e) determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof.

[0037] In any of the preceding embodiments, prior to the embedding, the biological sample can be a fresh frozen tissue sample or a decrosslinked tissue sample. In any of the preceding embodiments, prior to the embedding, the biological sample can be a fixed tissue sample, and between the embedding and removing, the biological sample embedded in the hydrogel matrix can be decrosslinked.

[0038] In any of the preceding embodiments, the target RNA can be 3’ tethered to the hydrogel matrix. In any of the preceding embodiments, the target RNA can be 5’ tethered to the hydrogel matrix.

[0039] In any of the preceding embodiments, the capture domain in the capture probe can comprise a 3’ poly(dT) sequence.

[0040] In any of the preceding embodiments, the method can comprise generating an extended capture probe comprising, from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released target RNA. In any of the preceding embodiments, the extended capture probe can further comprise a 3’ end homopolymer sequence. In any of the preceding embodiments, the method can further comprise hybridizing a template switch oligonucleotide to the extended capture probe, and extending the extended capture probe using the template switch oligonucleotide as a template to generate a further extended capture probe. In any of the preceding embodiments, the method can further comprise hybridizing a primer to the further extended capture probe at a sequence complementary to the template switch oligonucleotide, and extending the primer using the further extended capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a sequence of the released target RNA and a complementary sequence of the spatial barcode.

[0041] In any of the preceding embodiments, the spatially labeled polynucleotide or a portion thereof can be released from the further extended capture probe for analysis. In any of the preceding embodiments, the spatially labeled polynucleotide or a portion thereof released from the spatial array can be analyzed by nucleic acid sequencing.ny-2785575202412020440

[0042] In any of the preceding embodiments, the method can comprise imaging the spatial array using fluorescence microscopy. In any of the preceding embodiments, the method can comprise amplifying the released target RNA, the extended capture probe, the further extended capture probe, or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released target RNA, the extended capture probe, the further extended capture probe, or the spatially labeled polynucleotide, or an amplification product thereof. In any of the preceding embodiments, the method can comprise performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array.

[0043] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) hybridizing a probe that is substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; d) extending the hybridized probe to form an extended probe, wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe; e) releasing the extended probe from the hydrogel matrix; f) hybridizing or ligating the released extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and g) determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0044] In any of the preceding embodiments, the biological sample can be a fresh frozen tissue sample or a decrosslinked tissue sample. In any of the preceding embodiments, prior to the embedding, the biological sample can be a fixed tissue sample, and between the embedding and the removing, the biological sample embedded in the hydrogel matrix can be decrosslinked.

[0045] In any of the preceding embodiments, the target RNA can be 3’ tethered to the hydrogel matrix. In any of the preceding embodiments, the target RNA can be 5’ tethered to the hydrogel matrix.

[0046] In any of the preceding embodiments, the probe can hybridize to a poly(A) sequence in the target RNA tethered to the hydrogel matrix. In any of the precedingny-2785575202412020440 embodiments, the probe can comprise a 3’ poly(dT) sequence followed on the 5’ by a non- poly(dT) sequence. In any of the preceding embodiments, the holopolymer sequence can comprise a poly(C) sequence.

[0047] In any of the preceding embodiments, the capture domain in the capture probe can comprise a template switch oligonucleotide sequence and a homopolymer sequence complementary to the homopolymer sequence at the 3’ end of the extended probe.

[0048] In any of the preceding embodiments, the method can comprise extending the extended probe using the capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a complementary sequence of the released target RNA and a complementary sequence of the spatial barcode. In any of the preceding embodiments, the spatially labeled polynucleotide or a portion thereof can be released from the capture probe for analysis. In any of the preceding embodiments, the spatially labeled polynucleotide or portion thereof released from the spatial array can be analyzed by nucleic acid sequencing.

[0049] In any of the preceding embodiments, the method can comprise imaging the spatial array using fluorescence microscopy. In any of the preceding embodiments, the method can comprise amplifying the released target RNA, the extended probe, the capture probe, or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released target RNA, the extended probe, the capture probe, or the spatially labeled polynucleotide, or an amplification product thereof. In any of the preceding embodiments, the method can comprise performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array.

[0050] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) hybridizing a first probe that is substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; d) extending the hybridized probe to form an extended probe, wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe; e) hybridizing a second probe to the extended probe in the hydrogel matrix and further extending the extended probe using the second probe as a template to form a further extended probe, wherein the extension comprises incorporating a polyuridine-polyadenine double stranded region at the end of a duplex formedny-2785575202412020440 between the second probe and the further extended probe; f) releasing the further extended probe (e.g., the further extended probe comprising the double stranded region) from the hydrogel matrix; g) hybridizing or ligating the released further extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and h) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the further extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0051] In any of the preceding embodiments, the biological sample can be a fresh frozen tissue sample or a decrosslinked tissue sample. In any of the preceding embodiments, prior to the embedding, the biological sample can be a fixed tissue sample, and between the embedding and the removing, the biological sample embedded in the hydrogel matrix can be decrosslinked.

[0052] In any of the preceding embodiments, the target RNA can be 3’ tethered to the hydrogel matrix. In any of the preceding embodiments, the target RNA can be 5’ tethered to the hydrogel matrix.

[0053] In any of the preceding embodiments, the first probe can hybridize to a poly(A) sequence in the target RNA tethered to the hydrogel matrix. In any of the preceding embodiments, the first probe can comprise a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence. In any of the preceding embodiments, the second probe can comprise a 5’ poly(U) sequence, a template switch oligonucleotide sequence, and a homopolymer sequence complementary to the homopolymer sequence at the 3’ end of the extended probe. In any of the preceding embodiments, the further extended probe can comprise a 3’ poly(A) sequence.

[0054] In any of the preceding embodiments, the releasing can comprise releasing the tethered RNA from the hydrogel matrix. In any of the preceding embodiments, the releasing can comprise releasing the further extended probe from the target RNA, with or without cleaving or degrading the target RNA, and with or without releasing the target RNA from the hydrogel matrix. In any of the preceding embodiments, the releasing can comprise contacting the hydrogel matrix with an RNase H.

[0055] In any of the preceding embodiments, the capture domain in the capture probe can comprise a 3’ poly(dT) sequence.ny-2785575202412020440

[0056] In any of the preceding embodiments, the method can comprise generating a spatially labeled polynucleotide comprising: i) from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released further extended probe, or ii) from 5’ to 3’: a sequence of the released further extended probe and a complementary sequence of the spatial barcode.

[0057] In any of the preceding embodiments, the spatially labeled polynucleotide or a portion thereof can be released from the spatial array for analysis. In any of the preceding embodiments, the spatially labeled polynucleotide or a portion thereof released from the spatial array can be analyzed by nucleic acid sequencing.

[0058] In any of the preceding embodiments, the method can comprise imaging the spatial array using fluorescence microscopy. In any of the preceding embodiments, the method can comprise amplifying the released further extended probe or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released further extended probe or the spatially labeled polynucleotide or an amplification product thereof. In any of the preceding embodiments, the method can comprise performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array.

[0059] In any of the preceding embodiments, the biological sample can be contacted with a plurality of transposomes, wherein a transposome of the plurality of transposomes comprises (i) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, (ii) a linker, and (iii) a reactive group, thereby tagmenting an open chromatin in the biological sample with the plurality of transposomes, and wherein the tagmented open chromatin is tethered to the hydrogel matrix via the reactive group of the tagmented open chromatin, wherein after the biological sample is removed from the hydrogel matrix, the tagmented open chromatin is released from the hydrogel matrix, the released tagmented open chromatin is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the tagmented open chromatin or a complement thereof and (ii) the spatial barcode or a complement thereof is determined.

[0060] In any of the preceding embodiments, the biological sample can be contacted with an antibody that binds to a chromatin protein or chromatin-associated protein in theny-2785575202412020440 biological sample, and with a transposome-binding moiety complex that binds to the antibody, wherein the transposome-binding moiety complex comprises (i) a transposase, (ii) an antibody- binding moiety, (iii) a transposon end sequence comprising a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, and (iv) a reactive group, thereby tagmenting a genomic DNA in the biological sample with the transposome-binding moiety complex, wherein the tagmented genomic DNA is tethered to the hydrogel matrix via the reactive group of the transposome-binding moiety complex, wherein after the biological sample is removed from the hydrogel matrix, the tagmented genomic DNA is released from the hydrogel matrix, the released tagmented genomic DNA is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the tagmented genomic DNA or a complement thereof and (ii) the spatial barcode or a complement thereof is determined.

[0061] In any of the preceding embodiments, the biological sample can be contacted with an antibody-oligonucleotide conjugate, wherein the antibody-oligonucleotide conjugate comprises (i) an antibody that binds to a target protein in the biological sample, (ii) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, (iii) a barcode sequence associated with the antibody, and (iv) a reactive group, wherein the antibody-oligonucleotide conjugate is tethered to the hydrogel matrix via the reactive group of the antibody-oligonucleotide conjugate, wherein after the biological sample is removed from the hydrogel matrix, the antibody-oligonucleotide conjugate or a portion thereof is released from the hydrogel matrix, the capture sequence of the released antibody-oligonucleotide conjugate or portion thereof is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the barcode sequence associated with the antibody or a complement thereof and (ii) the spatial barcode or a complement thereof is determined.

[0062] In any of the preceding embodiments, two or more or all of the released ligation product, the released target RNA, the released extended probe, the released further extended probe, the released tagmented open chromatin, the released tagmented genomic DNA, the released antibody-oligonucleotide conjugate or portion thereof are hybridized or ligated to capture probes comprising capture domains of different sequences on the spatial array.ny-2785575202412020440

[0063] In some embodiments, disclosed herein is a method for analyzing a biological sample, comprising: a) contacting the biological sample with a plurality of transposomes, wherein a transposome of the plurality comprises (i) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, (ii) a linker, and (iii) a reactive group; b) tagmenting an open chromatin in the biological sample with the plurality of transposomes; c) embedding the biological sample in a hydrogel matrix, wherein the tagmented open chromatin is tethered to the hydrogel matrix via the reactive group of the tagmented open chromatin; d) removing the biological sample from the hydrogel matrix; e) releasing the tagmented open chromatin from the hydrogel matrix, wherein the released tagmented open chromatin is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and f) determining a profile of the open chromatin in the biological sample by determining the sequence of (i) the tagmented open chromatin or a complement thereof and (ii) the spatial barcode or a complement thereof.

[0064] In any of the preceding embodiments, the linker can comprise an antibody- binding moiety. In any of the preceding embodiments, the antibody-binding moiety can comprise protein A, protein G, or a fusion protein thereof. In any of the preceding embodiments, the capture sequence can be in a first transposon end sequence, and the transposome or the transposome-binding moiety complex can comprise a second transposon end sequence comprising a functional sequence. In any of the preceding embodiments, the functional sequence can comprise a primer binding sequence or a complement thereof.

[0065] In some embodiments, disclosed herein is a method for analyzing a biological sample, comprising: a) contacting the biological sample with: an antibody that binds to a chromatin protein or chromatin-associated protein in the biological sample, a transposome- binding moiety complex that binds to the antibody, wherein the transposome-binding moiety complex comprises (i) a transposase, (ii) an antibody-binding moiety, (iii) a transposon end sequence comprising a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, and (iv) a reactive group; b) tagmenting a genomic DNA in the biological sample with the transposome-binding moiety complex; c) embedding the biological sample in a hydrogel matrix, wherein the tagmented genomic DNA is tethered to the hydrogel matrix via the reactive group of the transposome-binding moiety complex; d) removingny-2785575202412020440 the biological sample from the hydrogel matrix; e) releasing the tagmented genomic DNA from the hydrogel matrix, wherein the released tagmented genomic DNA is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and f) determining the sequence of (i) the tagmented genomic DNA or a complement thereof and (ii) the spatial barcode or a complement thereof.

[0066] In any of the preceding embodiments, the chromatin protein or chromatin- associated protein can comprise a histone or a non-histone DNA binding protein. In any of the preceding embodiments, the non-histone DNA binding protein can comprise a transcription factor or a transcription co-factor. In any of the preceding embodiments, the antibody can bind to one or more epigenetic modifications. In any of the preceding embodiments, the antibody can bind to one or more histone modifications.

[0067] In any of the preceding embodiments, the antibody can directly bind to the chromatin protein or chromatin-associated protein. In any of the preceding embodiments, the antibody-binding moiety in the transposome-binding moiety complex can directly bind to the antibody.

[0068] In any of the preceding embodiments, the antibody can be a primary antibody that directly binds to the chromatin protein or chromatin-associated protein. In any of the preceding embodiments, the biological sample can be contacted with a secondary antibody that directly binds to the primary antibody. In any of the preceding embodiments, the antibody- binding moiety in the transposome-binding moiety complex can directly bind to the secondary antibody.

[0069] In some embodiments, disclosed herein is a method for analyzing a biological sample, comprising: a) contacting the biological sample with an antibody-oligonucleotide conjugate comprising (i) an antibody that binds to a target protein in the biological sample, (ii) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, (iii) a barcode sequence associated with the antibody, and (iv) a reactive group; b) embedding the biological sample in a hydrogel matrix, wherein the antibody- oligonucleotide conjugate is tethered to the hydrogel matrix via the reactive group of the antibody-oligonucleotide conjugate; c) removing the biological sample from the hydrogel matrix; d) releasing the antibody-oligonucleotide conjugate or a portion thereof from the hydrogel matrix, wherein capture sequence of the released antibody-oligonucleotide conjugate orny-2785575202412020440 portion thereof is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and e) determining the sequence of (i) the barcode sequence associated with the antibody or a complement thereof and (ii) the spatial barcode or a complement thereof.

[0070] In any of the preceding embodiments, the reactive group can comprise a 5’ acrylic phosphoramidite. In any of the preceding embodiments, the transposome, the transposome-binding moiety complex, and / or the antibody-oligonucleotide conjugate can comprise a cleavable linker connected to the reactive group. In any of the preceding embodiments, the transposome, the transposome-binding moiety complex, and / or the antibody- oligonucleotide conjugate can comprise a functional sequence. In any of the preceding embodiments, the functional sequence can comprise a primer binding sequence or a complement thereof.

[0071] Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The following drawings illustrate certain embodiments of the features and advantages of the present disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.

[0073] FIG. 1A shows an exemplary sandwiching process where a first substrate (e.g., a slide), including a biological sample such as a tissue sample attached thereto, and a second substrate (e.g., an array slide) are brought into proximity with one another.

[0074] FIG. 1B shows a fully formed sandwich configuration creating a chamber formed from the one or more spacers, the first substrate, and the second substrate.

[0075] FIG. 2A shows a perspective view of an exemplary sample handling apparatus in a closed position.ny-2785575202412020440

[0076] FIG. 2B shows a perspective view of an exemplary sample handling apparatus in an open position.

[0077] FIG. 3A shows the first substrate angled over (superior to) the second substrate.

[0078] FIG. 3B shows that as the first substrate lowers, and / or as the second substrate rises, the dropped side of the first substrate may contact a drop of reagent medium.

[0079] FIG. 3C shows a full closure of the sandwich between the first substrate and the second substrate with one or more spacers contacting both the first substrate and the second substrate.

[0080] FIG. 4A shows a side view of the angled closure workflow.

[0081] FIG. 4B shows a top view of the angled closure workflow.

[0082] FIG. 5 is a schematic diagram showing an example of a barcoded capture probe, as described herein.

[0083] FIG. 6 shows a schematic illustrating a cleavable capture probe.

[0084] FIG. 7 shows exemplary capture domains on capture probes.

[0085] FIG. 8 shows an exemplary arrangement of barcoded features within an array.

[0086] FIG. 9A shows and exemplary workflow for performing templated capture and producing a ligation product, and FIG. 9B shows an exemplary workflow for capturing a ligation product from FIG. 9A on a substrate.

[0087] FIG. 10 is a schematic diagram of an exemplary analyte capture agent.

[0088] FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1124 and an analyte capture agent 1126.

[0089] FIG. 12A provides an embodiment for spatial transcriptomic profiling of a sample (using FFPE tissue sections as an example) mediated by tethering of the 3’ or 5’ end of a target RNA molecule to a matrix such as a hydrogel matrix. A first probe and a second probe (e.g., RNA-templated ligation probes, hereinafter “RTL probes”) are hybridized to the tethered RNA and ligated to generate a ligation product. The ligation product can be captured on a capture array for subsequent analysis. FIG. 12B shows probe hybridization to the tethered target mRNA and probe ligation between the first and the second probes on the tethered target mRNA. FIG. 12C shows a schematic of a capture probe from a spatial array (e.g., a hydrogel spatialny-2785575202412020440 array) hybridized to the ligation product generated from ligating the first and second probes from FIG. 12B.

[0090] FIG. 13A provides an embodiment for spatial transcriptomic profiling of a sample (using fresh frozen tissue sections as an example) mediated by tethering of the 3’ or 5’ end of a target RNA molecule to a matrix (e.g. a hydrogel). FIG. 13B shows capture of a target mRNA and reverse transcription (RT) on a spatial array (e.g., a hydrogel barcoded capture array), using the 3’ poly(dT)VN as a primer to reverse transcribe the RNA sequence. FIG. 13C illustrates the template switch oligo (TSO) priming, transcript extension, second strand synthesis priming, and second strand synthesis steps to generate a spatially labeled polynucleotide.

[0091] FIG. 14A provides an embodiment for spatial transcriptomic profiling of a sample (using fresh frozen tissue sections as an example) mediated by tethering of the 3’ end of a target RNA molecule to a hydrogel and leveraging the TSO sequence as the capture sequence complementary to the capture domain of a capture probe on the spatial array. A probe is hybridized to the poly(A) of the tethered RNA as a primer for reverse transcription, followed by the TSO priming, template switch and transcript extension steps to generate a spatially labeled polynucleotide. FIG. 14B demonstrates reverse transcription of the tethered RNA using a poly(A) hybridized probe as a primer. FIG. 14C demonstrates template-switch oligo (TSO) priming, template switching, then transcript extension of the reverse-transcription product hybridized to the array.

[0092] FIG. 15A provides an embodiment for spatial transcriptomic profiling of a sample (using fresh frozen tissue sections as an example) mediated by tethering of the 3’ end of a target RNA molecule to a hydrogel, where first strand cDNA synthesis of mRNA tethered target is performed in the hydrogel, after which time the first strand cDNA is released and able to migrate to the capture probes on the array for hybridization. The reverse transcription and TSO priming steps are performed in the hydrogel, as shown in FIG. 15B, thereby generating a cDNA molecule comprising 3’ poly(A) for capture by a poly(dT) in a capture probe on a spatial array in order to generate a spatially labeled polynucleotide.

[0093] FIG. 16A provides an embodiment for combining spatial ATAC-seq and spatial transcriptomic profiling (RNA-seq) of a sample (using fresh frozen tissue sections as an example) mediated by tethering of the 3’ or 5’ end of a target RNA molecule to a hydrogel. FIG. 16B shows a modified transposon sequence comprising a PCR handle, a cleavable linker, and any-2785575202412020440 reactive group (e.g., 5’-acrydite). FIG. 16C shows the assembly of the transposon, specifically the configuration of the transposon ends, with a transposase, for example with Tn5, thereby generating a transposome capable of inserting the transposon ends into accessible genomic DNA. FIG. 16D shows an exemplary structure of transposed DNA tethered to a hydrogel matrix via the reactive group. FIG. 16E shows capturing a transposed DNA by a capture probe for the ATAC-seq modality and FIG. 16F shows capturing a target RNA by a capture probe for the RNA-seq modality, and the different capture probes can be combined on the array (e.g., in the same array spot, the capture probe molecules contain the same spatial barcode and different capture regions) for combined analysis.

[0094] FIG. 17A provides an embodiment of combined spatial ChIP-seq and spatial transcriptomic profiling (RNA-seq) of a sample (using fresh frozen tissue sections as an example) mediated by tethering of the 3’ or 5’ end of a target RNA molecule to a hydrogel. FIG. 17B shows a primary antibody (e.g., an antibody that binds to a chromatin protein or chromatin- associated protein), a secondary antibody that bidns to the primary antibody, and a transposome- binding moiety complex which comprises (i) a transposase (e.g., Tn5), (ii) an antibody-binding moiety (pA), and DNA adapter sequences. FIG. 17C depicts different ends of a transposon as they are combined with a transposase-pA complex to generate a transposome. FIG. 17D shows an exemplary structure of the transposed DNA tethered to a hydrogel matrix via the reactive group (e.g., 5’-acrydite).

[0095] FIG. 18A provides an embodiment for combining spatial protein analysis and spatial gene expression analysis using RNA-templated probe ligation in a sample (using FFPE tissue samples as an example) mediated by tethering of the 3’ or 5’ end of a target RNA molecule to a hydrogel. FIGS. 18B-18D each shows an example of an oligonucleotide-antibody conjugate for use in spatial protein analysis. FIG. 18E shows a capture probe configured to capture the capture sequence in the oligonucleotide-antibody conjugate shown in FIG. 18B and FIG. 18C, and a spatially labeled polynucleotide can be generated using hybridization followed by primer extension. FIG. 18F shows a capture probe configured to capture the capture sequence in the oligonucleotide-antibody conjugate shown in FIG. 18D, and a spatially labeled polynucleotide can be generated using a ligation / gap filing approach.

[0096] FIG. 19 shows an embodiment of spatial multiome analyte profiling mediated by tethering nucleic acid molecules to a hydrogel matrix and integrating different modalities ofny-2785575202412020440 spatial analysis by including capture probes comprising different capture regions on a spatially barcoded array.

[0097] FIG. 20A shows a sandwich arrangement for a boronic acid hydrogel-based reversible tethering scheme. FIG. 20B shows an exemplary electrophoretic migration of the target molecule to the capture array (e.g., hydrogel barcoded capture array). FIG. 20C depicts a thin film of indium tin oxide (ITO) deposited on both the sample substrate and the capture array. DETAILED DESCRIPTION

[0098] All publications, comprising patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0099] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0100] The terms “oligonucleotide,” “polynucleotide,” and “nucleic acid molecule,” used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term comprises, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, DNA- RNA combination molecules, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.

[0101] A “primer” used herein can be an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point ofny-2785575202412020440 initiation of nucleic acid synthesis and being extended from its 3' end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers are also used for polymerase chain reaction or other forms of amplification and extension. Primers usually are extended by a polymerase, for example a reverse transcriptase or a DNA polymerase.

[0102] “Ligation” refers to the formation of a covalent bond or linkage between the termini of two or more nucleic acids, e.g., oligonucleotides and / or polynucleotides, in a template-driven reaction. The nature of the bond or linkage may vary widely and the ligation may be carried out enzymatically or chemically. As used herein, ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5' carbon terminal nucleotide of one oligonucleotide with a 3' carbon of another nucleotide.

[0103] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein comprises (and describes) embodiments that are directed to that value or parameter per se.

[0104] As used herein, the singular forms “a,” “an,” and “the” comprise plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.”

[0105] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be comprised in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range comprises one or both of the limits, ranges excluding either or both of those comprisedny-2785575202412020440 limits are also comprised in the claimed subject matter. This applies regardless of the breadth of the range.

[0106] The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.

[0107] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, use of a), b), etc., or i), ii), etc. does not by itself connote any priority, precedence, or order of steps in the claims. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.

[0108] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0109] Particulars of the steps of the methods can be carried out as described herein, for example in Sections I and II; and / or using any suitable processes and methods for carrying out the particular steps. I. SPATIAL ANALYSIS METHODS

[0110] Spatial analysis methodologies described herein can provide a vast amount of analyte and / or expression data for a variety of analytes within a biological sample at high spatial resolution, while retaining native spatial context. Spatial analysis methods can include, e.g., the use of a capture probe including a spatial barcode (e.g., a nucleic acid sequence that provides information as to the location or position of an analyte within a cell or a tissue sample (e.g., mammalian cell or a mammalian tissue sample) and a capture domain that is capable of binding to an analyte (e.g., a protein and / or a nucleic acid) produced by and / or present in a cell. Spatial analysis methods and compositions can also include the use of a capture probe having a capture domain that captures an intermediate agent for indirect detection of an analyte. For example, the intermediate agent can include a nucleic acid sequence (e.g., a barcode) associated with theny-2785575202412020440 intermediate agent. Detection of the intermediate agent is therefore indicative of the analyte in the cell or tissue sample.

[0111] Non-limiting aspects of spatial analysis methodologies and compositions are described in U.S. Patent Nos. 11,447,807, 11,352,667, 11,168,350, 11,104,936, 11,008,608, 10,995,361, 10,913,975, 10,774,374, 10,724,078, 10,640,816, 10,494,662, 10,480,022, 10,364,457, 10,317,321, 10,059,990, 10,041,949, 10,030,261, 10,002,316, 9,879,313, 9,783,841, 9,727,810, 9,593,365, 8,951,726, 8,604,182, and 7,709,198; U.S. Patent Application Publication Nos. 2022 / 0010367, 2020 / 0239946, 2020 / 0080136, 2020 / 0277663, 2019 / 0330617, 2020 / 0256867, 2020 / 0224244, 2019 / 0085383, 2017 / 0241911, 2016 / 0108458, and 2013 / 0171621; PCT Publication Nos. WO2018 / 091676, WO2020 / 176788, WO2017 / 144338, and WO2016 / 057552; Non-patent literature references Rodriques et al., Science 363(6434):1463-1467, 2019; Lee et al., Nat. Protoc.10(3):442-458, 2015; Trejo et al., PLoS ONE 14(2):e0212031, 2019; Chen et al., Science 348(6233):aaa6090, 2015; Gao et al., BMC Biol. 15:50, 2017; and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018; the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022); and / or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022), both of which are available at the 10x Genomics Support Documentation website, and can be used herein in any combination, and each of which is incorporated herein by reference in their entireties. Further non-limiting aspects of spatial analysis methodologies and compositions are described herein.

[0112] Some general terminology that may be used in this disclosure can be found in Section (I)(b) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663 or US 2022 / 0010367 A1. Typically, a “barcode” is a label, or identifier, that conveys or is capable of conveying information (e.g., information about an analyte in a sample, a bead, and / or a capture probe). A barcode can be part of an analyte, or independent of an analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes. For the purpose of this disclosure, an “analyte” can include any biological substance, structure, moiety, or component to be analyzed. The term “target” can similarly refer to an analyte of interest.

[0113] Analytes can be broadly classified into one of two groups: nucleic acid analytes, and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but areny-2785575202412020440 not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitylation variants of proteins, sulfation variants of proteins, viral proteins (e.g., viral capsid, viral envelope, viral coat, viral accessory, viral glycoproteins, viral spike, etc.), extracellular and intracellular proteins, antibodies, and antigen binding fragments. In some embodiments, the analyte(s) can be localized to subcellular location(s), including, for example, organelles, e.g., mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, etc. In some embodiments, analyte(s) can be peptides or proteins, including without limitation antibodies and enzymes. Additional examples of analytes can be found in Section (I)(c) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. In some embodiments, an analyte can be detected indirectly, such as through detection of an intermediate agent, for example, a ligation product or an analyte capture agent (e.g., an oligonucleotide-conjugated antibody), such as those described herein.

[0114] A “biological sample” is typically obtained from the subject for analysis using any of a variety of techniques including, but not limited to, biopsy, surgery, and laser capture microscopy (LCM), and generally includes cells and / or other biological material from the subject. In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample (e.g., tissue sample) is a tissue microarray (TMA). A tissue microarray contains multiple representative tissue samples – which can be from different tissues or organisms – assembled on a single histologic slide. The TMA can therefore allow for high throughput analysis of multiple specimens at the same time. Tissue microarrays are paraffin blocks produced by extracting cylindrical tissue cores from different paraffin donor blocks and re-embedding these into a single recipient (microarray) block at defined array coordinates.

[0115] The biological sample as used herein can be any suitable biological sample described herein or known in the art. In some embodiments, the biological sample is a tissue sample. In some embodiments, the tissue sample is a solid tissue sample. In some embodiments, the biological sample is a tissue section (e.g., a fixed tissue section). In some embodiments, the tissue is flash-frozen and sectioned. Any suitable method described herein or known in the art can be used to flash-freeze and section the tissue sample. In some embodiments, the biologicalny-2785575202412020440 sample, e.g., the tissue, is flash-frozen using liquid nitrogen before sectioning. In some embodiments, the biological sample, e.g., a tissue sample, is flash-frozen using nitrogen (e.g., liquid nitrogen), isopentane, or hexane.

[0116] In some embodiments, the biological sample, e.g., the tissue, is embedded in a matrix e.g., optimal cutting temperature (OCT) compound to facilitate sectioning. OCT compound is a formulation of clear, water-soluble glycols and resins, providing a solid matrix to encapsulate biological (e.g., tissue) specimens. In some embodiments, the sectioning is performed by cryosectioning, for example using a microtome . In some embodiments, the methods further comprise a thawing step, after the cryosectioning.

[0117] The biological sample can be from a mammal. In some instances, the biological sample is from a human, mouse, or rat. In addition to the subjects described above, the biological sample can be obtained from non-mammalian organisms (e.g., a plants, an insect, an arachnid, a nematode (e.g., Caenorhabditis elegans), a fungi, an amphibian, or a fish (e.g., zebrafish)). A biological sample can be obtained from a prokaryote such as a bacterium, e.g., Escherichia coli, Staphylococci or Mycoplasma pneumoniae; an archaea; a virus such as Hepatitis C virus or human immunodeficiency virus; or a viroid. A biological sample can be obtained from a eukaryote, such as a patient derived organoid (PDO) or patient derived xenograft (PDX). The biological sample can include organoids, a miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy. Organoids can be generated from one or more cells from a tissue, embryonic stem cells, and / or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self- renewal and differentiation capacities. In some embodiments, an organoid is a cerebral organoid, an intestinal organoid, a stomach organoid, a lingual organoid, a thyroid organoid, a thymic organoid, a testicular organoid, a hepatic organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid. Subjects from which biological samples can be obtained can be healthy or asymptomatic individuals, individuals that have or are suspected of having a disease (e.g., cancer) or a pre- disposition to a disease, and / or individuals that are in need of therapy or suspected of needing therapy.ny-2785575202412020440

[0118] Biological samples can be derived from a homogeneous culture or population of the subjects or organisms mentioned herein or alternatively from a collection of several different organisms, for example, in a community or ecosystem.

[0119] Biological samples can include one or more diseased cells. A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines, or obtained as circulating tumor cells.

[0120] In some embodiments, the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, for example methanol. In some embodiments, instead of methanol, acetone, or an acetone-methanol mixture can be used. In some embodiments, the fixation is performed after sectioning. In some instances, when the biological sample is fixed with a fixative including an alcohol (e.g., methanol or acetone-methanol mixture), it is not decrosslinked afterward. In some preferred embodiments, the biological sample is fixed with a fixative including an alcohol (e.g., methanol or an acetone-methanol mixture) after freezing and / or sectioning. In some instances, the biological sample is flash-frozen, and then the biological sample is sectioned and fixed (e.g., using methanol, acetone, or an acetone-methanol mixture). In some instances when methanol, acetone, or an acetone-methanol mixture is used to fix the biological sample, the sample is not decrosslinked at a later step. In instances when the biological sample is frozen (e.g., flash frozen using liquid nitrogen and embedded in OCT) followed by sectioning and alcohol (e.g., methanol, acetone-methanol) fixation or acetone fixation, the biological sample is referred to as “fresh frozen”. In some embodiments, fixation of the biological sample e.g., using acetone and / or alcohol (e.g., methanol, acetone-methanol) is performed while the sample is mounted on a substrate (e.g., glass slide, such as a positively charged glass slide).

[0121] In some embodiments, the biological sample, e.g., the tissue sample, is fixed e.g., immediately after being harvested from a subject. In such embodiments, the fixative is preferably an aldehyde fixative, such as paraformaldehyde (PFA) or formalin. In some embodiments, the fixative induces crosslinks within the biological sample. In some embodiments, after fixing e.g., by formalin or PFA, the biological sample is dehydrated via sucrose gradient. In some instances, the fixed biological sample is treated with a sucrose gradient 27ny-2785575202412020440 and then embedded in a matrix e.g., OCT compound. In some instances, the fixed biological sample is not treated with a sucrose gradient, but rather is embedded in a matrix e.g., OCT compound after fixation. In some embodiments when a fixed frozen tissue sample is treated with a sucrose gradient, it can be rehydrated with an ethanol gradient. In some embodiments, the PFA or formalin fixed biological sample, which can be optionally dehydrated via sucrose gradient and / or embedded in OCT compound, is then frozen e.g., for storage or shipment. In such instances, the biological sample is referred to as “fixed frozen”. In preferred embodiments, a fixed frozen biological sample is not treated with methanol. In preferred embodiments, a fixed frozen biological sample is not paraffin embedded. Thus, in preferred embodiments, a fixed frozen biological sample is not deparaffinized. In some embodiments, a fixed frozen biological sample is rehydrated in an ethanol gradient.

[0122] In some instances, the biological sample (e.g., a fixed frozen tissue sample) is treated with a citrate buffer. Citrate buffer can be used for antigen retrieval to decrosslink antigens and fixation medium in the biological sample. Thus, any suitable decrosslinking agent can be used in addition to or alternatively to citrate buffer. In some embodiments, for example, the biological sample (e.g., a fixed frozen tissue sample) is decrosslinked with TE buffer.

[0123] In any of the foregoing, the biological sample can further be stained, imaged, and / or destained. For example, in some embodiments, a fresh frozen tissue sample or fixed frozen tissue sample is stained (e.g., via eosin and / or hematoxylin), imaged, destained (e.g., via HCl), or a combination thereof. In some embodiments, when a fresh frozen tissue sample is fixed in methanol, it is treated with isopropanol prior to being stained (e.g., via eosin and / or hematoxylin), imaged, destained (e.g., via HCl), or a combination thereof. In some embodiments when a fixed frozen tissue sample is treated with a sucrose gradient, it can be rehydrated with an ethanol gradient before being stained, (e.g., via eosin and / or hematoxylin), imaged, destained (e.g., via HCl), decrosslinked (e.g., via TE buffer or citrate buffer), or a combination thereof. In some embodiments, the biological sample can undergo further fixation (e.g., while mounted on a substrate), stained, imaged, and / or destained. For example, a fixed frozen biological sample may be subject to an additional fixing step (e.g., using PFA) before optional ethanol rehydration, staining, imaging, and / or destaining.

[0124] In any of the foregoing, the biological sample can be fixed using PAXgene. For example, the biological sample can be fixed using PAXgene in addition, or alternatively to, any-2785575202412020440 fixative disclosed herein or known in the art (e.g., alcohol, acetone, acetone-alcohol, formalin, paraformaldehyde). PAXgene is a non-cross-linking mixture of different alcohols, acid and a soluble organic compound that preserves morphology and bio-molecules. It is a two-reagent fixative system in which tissue is firstly fixed in a solution containing methanol and acetic acid then stabilized in a solution containing ethanol. See, Ergin B. et al., J Proteome Res. 2010 Oct 1;9(10):5188-96; Kap M. et al., PLoS One.; 6(11):e27704 (2011); and Mathieson W. et al., Am J Clin Pathol.; 146(1):25-40 (2016), all of which are herein incorporated by reference in their entireties, for a description and evaluation of PAXgene for tissue fixation. Thus, in some embodiments, when the biological sample, e.g., the tissue sample, is fixed in a fixative including alcohol, the fixative is PAXgene. In some embodiments, a fresh frozen tissue sample is fixed with PAXgene. In some embodiments, a fixed frozen tissue sample is fixed with PAXgene.

[0125] In some embodiments, the biological sample, e.g., the tissue sample is fixed, for example in methanol, acetone, acetone-methanol, PFA, PAXgene or is formalin-fixed and paraffin-embedded (FFPE). In some embodiments, the biological sample comprises intact cells. In some embodiments, the biological sample is a cell pellet, e.g., a fixed cell pellet, e.g., an FFPE cell pellet. FFPE samples are used in some instances in the RNA-templated ligation methods disclosed herein. A limitation of direct RNA capture for fixed samples is that the RNA integrity of fixed (e.g., FFPE) samples can be lower than a fresh sample, thereby making it more difficult to capture RNA directly, e.g., by capture of a common sequence such as a poly(A) tail of an mRNA molecule. However, by utilizing RTL probes that hybridize to RNA target sequences in the transcriptome, one can avoid a requirement for RNA analytes to have both a poly(A) tail and target sequences intact. Accordingly, RTL probes can be utilized to beneficially improve capture and spatial analysis of fixed samples. The biological sample, e.g., tissue sample, can be stained, and imaged prior, during, and / or after each step of the methods described herein. Any of the methods described herein or known in the art can be used to stain and / or image the biological sample. In some embodiments, the imaging occurs prior to destaining the sample. In some embodiments, the biological sample is stained using an H&E staining method. In some embodiments, the tissue sample is stained and imaged for about 10 minutes to about 2 hours (or any of the subranges of this range described herein). Additional time may be needed for staining and imaging of different types of biological samples.ny-2785575202412020440

[0126] The tissue sample can be obtained from any suitable location in a tissue or organ of a subject, e.g., a human subject. In some instances, the sample is a mouse sample. In some instances, the sample is a human sample. In some embodiments, the sample can be derived from skin, brain, breast, lung, liver, kidney, prostate, tonsil, thymus, testes, bone, lymph node, ovary, eye, heart, or spleen. In some instances, the sample is a human or mouse breast tissue sample. In some instances, the sample is a human or mouse brain tissue sample. In some instances, the sample is a human or mouse lung tissue sample. In some instances, the sample is a human or mouse tonsil tissue sample. In some instances, the sample is a human or mouse liver tissue sample. In some instances, the sample is a human or mouse bone, skin, kidney, thymus, testes, or prostate tissue sample. In some embodiments, the tissue sample is derived from normal or diseased tissue. In some embodiments, the sample is an embryo sample. The embryo sample can be a non-human embryo sample. In some instances, the sample is a mouse embryo sample.

[0127] Biological samples are also described in Section (I)(d) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0128] The following embodiments can be used with any of the methods described herein. In some embodiments, the biological sample (e.g., a fixed and / or stained biological sample) is imaged. In some embodiments, the biological sample is visualized or imaged using bright field microscopy. In some embodiments, the biological sample is visualized or imaged using fluorescence microscopy. Additional methods of visualization and imaging are known in the art. Non-limiting examples of visualization and imaging include expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflection microscopy, interference microscopy and confocal microscopy. In some embodiments, the sample is stained and imaged prior to adding reagents for analyzing captured analytes as disclosed herein to the biological sample.

[0129] In some embodiments, the methods include staining the biological sample. In some embodiments, the staining includes the use of hematoxylin and / or eosin. Non-limiting examples of stains include histological stains (e.g., hematoxylin and / or eosin) and immunological stains (e.g., fluorescent stains). In some embodiments, a biological sample can be stained using any number of biological stains, including but not limited to, acridine orange, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nileny-2785575202412020440 blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. In some instances, the biological sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner’s, Leishman, Masson’s trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright’s, and / or Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation.

[0130] In some embodiments, the staining includes the use of a detectable label selected from the group consisting of a radioisotope, a fluorophore, a chemiluminescent compound, a bioluminescent compound, or a combination thereof.

[0131] In some embodiments, a biological sample is permeabilized with one or more permeabilization reagents. For example, permeabilization of a biological sample can facilitate analyte capture. Exemplary permeabilization agents and conditions are described in Section (I)(d)(ii)(13) or the Exemplary Embodiments Section of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Briefly, in any of the methods described herein, the method includes a step of permeabilizing the biological sample. For example, the biological sample can be permeabilized to facilitate transfer of the extension products to the capture probes on the array. In some embodiments, the permeabilizing includes the use of an organic solvent (e.g., acetone, ethanol, and methanol), a detergent (e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), an enzyme (an endopeptidase, an exopeptidase, a protease), or combinations thereof. In some embodiments, the permeabilizing includes the use of an endopeptidase, a protease, SDS, polyethylene glycol tert-octylphenyl ether, polysorbate 80, and polysorbate 20, N-lauroylsarcosine sodium salt solution, saponin, Triton X-100™, Tween-20™, or combinations thereof. In some embodiments, the endopeptidase is pepsin. In some embodiments, the endopeptidase is Proteinase K. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63-66, 2010, the entire contents of which are incorporated herein by reference.

[0132] Array-based spatial analysis methods can involve the transfer of one or more analytes or derivatives thereof or proxies thereof from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes includes determining the identity of the analytes and the spatial location of the analytes within the biological sample. The spatial location of an analyte within the biological sample is determined based on the feature to which the analyte isny-2785575202412020440 bound (e.g., directly or indirectly) on the array, and the feature’s relative spatial location within the array.

[0133] A “capture probe” refers to any molecule capable of capturing (directly or indirectly) and / or labeling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe includes a barcode (e.g., a spatial barcode and / or a unique molecular identifier (UMI) and a capture domain). In some instances, the capture probe includes a homopolymer sequence, such as a poly(T) sequence. In some embodiments, a capture probe can include a cleavage domain and / or a functional domain (e.g., a primer-binding site, such as for next- generation sequencing (NGS)). See, e.g., Section (II)(b) (e.g., subsections (i)-(vi)) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. Generation of capture probes can be achieved by any appropriate method, including those described in Section (II)(d)(ii) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0134] In some instances, a capture probe and a nucleic acid analyte (or any other nucleic acid to nucleic acid interaction) occurs because the sequences of the two nucleic acids are substantially complementary to one another. By “substantial,” “substantially” and the like, two nucleic acid sequences can be complementary when at least 60% of the nucleotide residues of one nucleic acid sequence are complementary to nucleotide residues in the other nucleic acid sequence. The complementary residues within a particular complementary nucleic acid sequence need not always be contiguous with each other, and can be interrupted by one or more non- complementary residues within the complementary nucleic acid sequence. In some embodiments, at least 60%, but less than 100%, of the residues of one of the two complementary nucleic acid sequences are complementary to residues in the other nucleic acid sequence. In some embodiments, at least 70%, 80%, 90%, 95% or 99% of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence. Sequences are said to be “substantially complementary” when at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the residues of one nucleic acid sequence are complementary to residues in the other nucleic acid sequence. In some embodiments, the biological sample is mounted on a first substrate and the substrate comprising the array of capture probes is a second substrate. During this process, one or more analytes or analyte derivatives (e.g., intermediate agents; e.g., ligationny-2785575202412020440 products) are released from the biological sample and migrate to the second substrate comprising an array of capture probes. In some embodiments, the release and migration of the analytes or analyte derivatives to the second substrate comprising the array of capture probes occurs in a manner that preserves the original spatial context of the analytes in the biological sample. This method can be referred to as a sandwiching process, which is described e.g., in U.S. Patent Application Pub. No. 2021 / 0189475 and PCT Pub. Nos. WO 2021 / 252747 A1, WO 2022 / 061152 A2, and WO 2022 / 140028 A1.

[0135] In some aspects, the methods provided herein comprise use of an array of capture probes. In some aspects, the array of capture probes is generated by attaching oligonucleotides (e.g. a barcode) to a substrate. Oligonucleotides may be attached to the substrate according to the methods set forth in U.S. Patent Nos. 6,737,236, 7,259,258, 7,375,234, 7,309,593, 7,427,678, 5,610,287, 5,807,522, 5,837,860, and 5,472,881; U.S. Patent Application Publication Nos. 2008 / 0280773, 2011 / 0143967, and 2011 / 0059865; Shalon et al. (1996) Genome Research, 639– 645; Rogers et al. (1999) Analytical Biochemistry 266, 23–30; Stimpson et al. (1995) Proc. Natl. Acad. Sci. USA 92, 6379–6383; Beattie et al. (1995) Clin. Chem. 45, 700–706; Lamture et al. (1994) Nucleic Acids Research 22, 2121–2125; Beier et al. (1999) Nucleic Acids Research 27, 1970–1977; Joos et al. (1997) Analytical Biochemistry 247, 96–101; Nikiforov et al. (1995) Analytical Biochemistry 227, 201–209; Timofeev et al. (1996) Nucleic Acids Research 24, 3142–3148; Chrisey et al. (1996) Nucleic Acids Research 24, 3031–3039; Guo et al. (1994) Nucleic Acids Research 22, 5456–5465; Running and Urdea (1990) BioTechniques 8, 276–279; Fahy et al. (1993) Nucleic Acids Research 21, 1819–1826; Zhang et al. (1991) 19, 3929–3933; and Rogers et al. (1997) Gene Therapy 4, 1387–1392. The entire contents of each of the foregoing documents are incorporated herein by reference.

[0136] In some embodiments, oligonucleotides may be immobilized by spotting (e.g., DNA printing) on a substrate with reactive surface chemistry, such as a polymer (e.g., a hydrophilic polymer) containing epoxy reactive groups. In some embodiments, the polymer comprises a passivating polymer. In some embodiments, the polymer comprises a photoreactive group for attachment to the substrate (such as a glass slide). In some embodiments, the oligonucleotides may be immobilized in a DNA printing buffer, optionally wherein the printing buffer comprises a surfactant such as sarcosyl (e.g., a buffer containing sodium phosphate and about 0.06% sarcosyl). In some embodiments, after immobilization of the oligonucleotides, one or more washny-2785575202412020440 and / or blocking steps are performed. Blocking steps can comprise contacting the substrate with a solution that deactivates or blocks unreacted functional groups on the substrate surface. In one example, the blocking buffer can comprise ethanolamine (e.g., to deactivate epoxy silane or other epoxy reactive functional groups).

[0137] Arrays can be prepared by a variety of methods. In some embodiments, arrays are prepared through the synthesis (e.g., in situ synthesis) of oligonucleotides on the array, or by jet printing or lithography. For example, light-directed synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid-phase DNA synthesis. To implement photolithographic synthesis, synthetic linkers modified with photochemical protecting groups can be attached to a substrate and the photochemical protecting groups can be modified using a photolithographic mask (applied to specific areas of the substrate) and light, thereby producing an array having localized photo-deprotection. Many of these methods are known in the art, and are described e.g., in Miller et al., “Basic concepts of microarrays and potential applications in clinical microbiology.” Clinical microbiology reviews 22.4 (2009): 611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676, the contents of each of which are incorporated herein by reference in their entirety.

[0138] In some embodiments, a substrate comprising an array of molecules is provided, e.g., in the form of a lawn of polymers (e.g., oligonucleotides), or polymers on the substrate in a pre- determined pattern. Examples of polymers on an array may include, but are not limited to, nucleic acids, peptides, phospholipids, polysaccharides, heteromacromolecules in which a known drug is covalently bound to any of the above, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates. The molecules occupying different features of an array typically differ from one another, although some redundancy in which the same polymer occupies multiple features can be useful as a control. For example, in a nucleic acid array, the nucleic acid molecules within the same feature are typically the same, whereas nucleic acid molecules occupying different features are mostly different from one another.

[0139] In some examples, the molecules on the array may be nucleic acids. The nucleic acid molecule can be single-stranded or double-stranded. Nucleic acid molecules on an array may be DNA or RNA. The DNA may be single-stranded or double-stranded. The DNA may include, but are not limited to, mitochondrial DNA, cell-free DNA, complementary DNA (cDNA), genomicny-2785575202412020440 DNA, plasmid DNA, cosmid DNA, bacterial artificial chromosome (BAC), or yeast artificial chromosome (YAC). The RNA may include, but are not limited to, mRNAs, tRNAs, snRNAs, rRNAs, retroviruses, small non-coding RNAs, microRNAs, polysomal RNAs, pre-mRNAs, intronic RNA, viral RNA, cell free RNA and fragments thereof. The non-coding RNA, or ncRNA can include snoRNAs, microRNAs, siRNAs, piRNAs and long non-coding RNAs (lncRNAs).

[0140] In some embodiments, the molecules on an array comprise oligonucleotide barcodes. A barcode sequence can be of varied length. In some embodiments, the barcode sequence is about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, or about 70 nucleotides in length. In some embodiments, the barcode sequence is between about 4 and about 25 nucleotides in length. In some embodiments, the barcode sequences is between about 10 and about 50 nucleotides in length. The nucleotides can be completely contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by 1 or more nucleotides. In some embodiments, the barcode sequence can be about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 nucleotides or longer. In some embodiments, the barcode sequence can be at least about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 nucleotides or longer. In some embodiments, the barcode sequence can be at most about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25 nucleotides or shorter.

[0141] The oligonucleotide can include one or more (e.g., two or more, three or more, four or more, five or more) Unique Molecular Identifiers (UMIs). A unique molecular identifier is a contiguous nucleic acid segment or two or more non-contiguous nucleic acid segments that function as a label or identifier for a particular analyte, or for a capture probe that binds a particular analyte (e.g., via the capture domain).ny-2785575202412020440

[0142] A UMI can be unique. A UMI can include one or more specific polynucleotides sequences, one or more random nucleic acid and / or amino acid sequences, and / or one or more synthetic nucleic acid and / or amino acid sequences.

[0143] In some embodiments, the UMI is a nucleic acid sequence that does not substantially hybridize to analyte nucleic acid molecules in a biological sample. In some embodiments, the UMI has less than 90% sequence identity (e.g., less than 80%, 70%, 60%, 50%, or less than 40% sequence identity) to the nucleic acid sequences across a substantial part (e.g., 80% or more) of the nucleic acid molecules in the biological sample.

[0144] The UMI can include from about 6 to about 20 or more nucleotides within the sequence of capture probes, e.g., barcoded oligonucleotides in an array generated using a method disclosed herein. In some embodiments, the length of a UMI sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of a UMI sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of a UMI sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter. These nucleotides can be contiguous, i.e., in a single stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences that are separated by 1 or more nucleotides. Separated UMI subsequences can be from about 4 to about 16 nucleotides in length. In some embodiments, the UMI subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0145] In some embodiments, a UMI is attached to other parts of the oligonucleotide in a reversible or irreversible manner. In some embodiments, a UMI is added to, for example, a fragment of a DNA or RNA sample before sequencing of the analyte. In some embodiments, a UMI allows for identification and / or quantification of individual sequencing-reads. In some embodiments, a UMI is used as a fluorescent barcode for which fluorescently labeled oligonucleotide probes hybridize to the UMI.

[0146] In some embodiments, the features on the substrate may correspond to regions of a substrate in which one or more barcodes have been incorporated. In some embodiments, the feature(s) may be about 0.5 micron, about 1 micron, about 2.5 microns, about 5 microns, aboutny-2785575202412020440 10 microns, about 15 microns, about 20 microns, about 25 microns, about 30 microns, about 35 microns, about 40 microns, about 45 microns, about 50 microns, about 55 microns, about 60 microns, about 65 microns, about 70 microns, about 75 microns, about 80 microns, about 85 microns, about 90 microns, about 100 microns or greater in diameter. In some embodiments, the feature(s) may be no more than 0.5 micron, no more than 1 micron, no more than 2.5 microns, no more than 5 microns, no more than 10 microns, no more than 15 microns, no more than 20 microns, no more than 25 microns, no more than 30 microns, no more than 35 microns, no more than 40 microns, no more than 45 microns, no more than 50 microns, no more than 55 microns, no more than 60 microns, no more than 65 microns, no more than 70 microns, no more than 75 microns, no more than 80 microns, no more than 85 microns, no more than 90 microns, or no more than 100 microns in diameter. In some embodiments, the features on the substrate are below 10 microns in diameter (e.g., single cell scale resolution) and provide high throughput readout (e.g., by sequencing) for analyzing a sample, such as a tissue sample. In some embodiments, the features on the substrate comprise barcoded squares and the substrate comprises a continuous grid of squares comprising spatially barcoded oligonucleotides in each square. In some embodiments, the features on the substrate comprise one or more 1x1 µm squares, one or more 2x2 µm squares, one or more 3x3 µm squares, one or more 4x4 µm squares, one or more 5x5 µm squares, one or more 6x6 µm squares, one or more 7x7 µm squares, one or more 8x8 µm squares, one or more 9x9 µm squares, and / or one or more 10x10 µm squares. In some embodiments, the features on the substrate comprise 2x2 µm squares. Methods for generating features on a substrate are disclosed, for example, in US 2022 / 0228201 A1, US 2022 / 0314187 A1, US 2022 / 0228210 A1, US 2024 / 0076721 A1, US 2024 / 0002932 A1, US 2024 / 0076722 A1, US 2024 / 0026444 A1, US 2024 / 0060127 A1, US 2024 / 0084359 A1, US 2024 / 0117338 A1, US 2024 / 0076656 A1, and US 2024 / 0167077 A1, all of which are herein incorporated by reference in their entireties.

[0147] FIG. 1A shows an exemplary sandwiching process 100 where a first substrate (e.g., slide 103), including a biological sample 102, and a second substrate (e.g., slide 104 including an array 106 having spatially barcoded capture probes) are brought into proximity with one another. As shown in FIG. 1A a liquid reagent drop (e.g., permeabilization solution 105) is introduced on the second substrate in proximity to the capture probes 106 and in between the biological sample 102 and the second substrate (e.g., slide 104 including an array 106 havingny-2785575202412020440 spatially barcoded capture probes). The permeabilization solution 105 may release analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) that can be captured by the capture probes of the array 106.

[0148] During the exemplary sandwiching process, the first substrate is aligned with the second substrate, such that at least a portion of the biological sample is aligned with at least a portion of the capture probes (e.g., aligned in a sandwich configuration). As shown, the second substrate (e.g., array slide 104) is in an inferior position to the first substrate (e.g., slide 103). In some embodiments, the first substrate (e.g., slide 103) may be positioned superior to the second substrate (e.g., slide 104). A reagent medium 105 within a gap between the first substrate (e.g., slide 103) and the second substrate (e.g., slide 104) creates a liquid interface between the two substrates. The reagent medium may be a permeabilization solution which permeabilizes and / or digests the biological sample 102. In some embodiments wherein the biological sample 102 has been pre-permeabilized, the reagent medium is not a permeabilization solution. Herein, the reagent medium may also comprise one or more of a monovalent salt, a divalent salt, ethylene carbonate, and / or glycerol. In some embodiments, analytes (e.g., mRNA transcripts) and / or analyte derivatives (e.g., intermediate agents; e.g., ligation products) of the biological sample 102 may release from the biological sample, and actively or passively migrate (e.g., diffuse) across the gap toward the capture probes on the array 106. Alternatively, in certain embodiments, migration of the analyte or analyte derivative (e.g., intermediate agent; e.g., ligation product) from the biological sample is performed actively (e.g., electrophoretic, by applying an electric field to promote migration). Exemplary methods of electrophoretic migration are described in WO 2020 / 176788, and US. Patent Application Pub. No. 2021 / 0189475, all of which is herein incorporated by reference in their entireties.

[0149] As further shown, one or more spacers 110 may be positioned between the first substrate (e.g., slide 103) and the second substrate (e.g., array slide 104 including array 106 comprising spatially barcoded capture probesforming array features such as array spots located on the slide). The one or more spacers 110 may be configured to maintain a separation distance between the first substrate and the second substrate. While the one or more spacers 110 is shown as disposed on the second substrate, the spacer may additionally or alternatively be disposed on the first substrate.ny-2785575202412020440

[0150] In some embodiments, the one or more spacers 110 is configured to maintain a separation distance between first and second substrates that is between about 2 microns and 1 mm (e.g., between about 2 microns and 800 microns, between about 2 microns and 700 microns, between about 2 microns and 600 microns, between about 2 microns and 500 microns, between about 2 microns and 400 microns, between about 2 microns and 300 microns, between about 2 microns and 200 microns, between about 2 microns and 100 microns, between about 2 microns and 25 microns, or between about 2 microns and 10 microns), measured in a direction orthogonal to the surface of first substrate that supports the biological sample. In some instances, the separation distance is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 microns. In some embodiments, the separation distance is less than 50 microns. In some embodiments, the separation distance is less than 25 microns. In some embodiments, the separation distance is less than 20 microns. The separation distance may include a distance of at least 2 µm.

[0151] FIG. 1B shows a fully formed sandwich configuration 125 creating a chamber 150 formed from the one or more spacers 110, the first substrate (e.g., the slide 103), and the second substrate (e.g., the slide 104 including an array 106 having spatially barcoded capture probes) in accordance with some example implementations. In the example of FIG. 1B, the liquid reagent (e.g., the permeabilization solution 105) fills the volume of the chamber 150 and may create a permeabilization buffer that allows analytes (e.g., mRNA transcripts and / or other molecules) or analyte derivatives (e.g., intermediate agents; e.g., ligation products) to diffuse from the biological sample 102 toward the capture probes of the second substrate (e.g., slide 104). In some aspects, flow of the permeabilization buffer may deflect transcripts and / or molecules from the biological sample 102 and may affect diffusive transfer of analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) for spatial analysis. A partially or fully sealed chamber 150 resulting from the one or more spacers 110, the first substrate, and the second substrate may reduce or prevent flow from undesirable convective movement of transcripts and / or molecules over the diffusive transfer from the biological sample 102 to the capture probes.

[0152] The sandwiching process methods described above can be implemented using a variety of hardware components. For example, the sandwiching process methods can be implemented using a sample holder (also referred to herein as a support device, a sampleny-2785575202412020440 handling apparatus, and an array alignment device). Further details on support devices, sample holders, sample handling apparatuses, or systems for implementing a sandwiching process are described in, e.g., US. Patent Application Pub. Nos. 2021 / 0189475 and 2024 / 0033743, all of which are herein incorporated by reference in their entireties.

[0153] In some embodiments of a sample holder, the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate comprising a biological sample. The first retaining mechanism can be configured to retain the first substrate disposed in a first plane. The sample holder can further include a second member including a second retaining mechanism configured to retain a second substrate disposed in a second plane. The sample holder can further include an alignment mechanism connected to one or both of the first member and the second member. The alignment mechanism can be configured to align the first and second members along the first plane and / or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane. The adjustment mechanism may be configured to move the second member along the axis orthogonal to the second plane and / or move the first member along an axis orthogonal to the first plane.

[0154] In some embodiments, the adjustment mechanism includes a linear actuator. In some embodiments, the linear actuator is configured to move the second member along an axis orthogonal to the plane of the first member and / or the second member. In some embodiments, the linear actuator is configured to move the first member along an axis orthogonal to the plane of the first member and / or the second member. In some embodiments, the linear actuator is configured to move the first member, the second member, or both the first member and the second member at a velocity of at least 0.1 mm / sec. In some embodiments, the linear actuator is configured to move the first member, the second member, or both the first member and the second member with an amount of force of at least 0.1 lbs.

[0155] FIG. 2A is a perspective view of an example sample handling apparatus 200 in a closed position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes a first member 204, a second member 210, optionally an image capture device 220, a first substrate 206, optionally a hinge 215, and optionally a mirror 216. The hinge 215 may be configured to allow the first member 204 to be positioned in an open or closedny-2785575202412020440 configuration by opening and / or closing the first member 204 in a clamshell manner along the hinge 215.

[0156] FIG. 2B is a perspective view of the example sample handling apparatus 200 in an open position in accordance with some example implementations. As shown, the sample handling apparatus 200 includes one or more first retaining mechanisms 208 configured to retain one or more first substrates 206. In the example of FIG. 2B, the first member 204 is configured to retain two first substrates 206, however the first member 204 may be configured to retain more or fewer first substrates 206.

[0157] In some aspects, when the sample handling apparatus 200 is in an open position (e.g., in FIG. 2B), the first substrate 206 and / or the second substrate 212 may be loaded and positioned within the sample handling apparatus 200 such as within the first member 204 and the second member 210, respectively. As noted, the hinge 215 may allow the first member 204 to close over the second member 210 and form a sandwich configuration.

[0158] In some aspects, after the first member 204 closes over the second member 210, an adjustment mechanism of the sample handling apparatus 200 may actuate the first member 204 and / or the second member 210 to form the sandwich configuration for the permeabilization step (e.g., bringing the first substrate 206 and the second substrate 212 closer to each other and within a threshold distance for the sandwich configuration). The adjustment mechanism may be configured to control a speed, an angle, a force, or the like of the sandwich configuration.

[0159] In some embodiments, the biological sample (e.g., sample 102 from FIG. 1A) may be aligned within the first member 204 (e.g., via the first retaining mechanism 208) prior to closing the first member 204 such that a desired region of interest of the sample is aligned with the barcoded array of the second substrate (e.g., the slide 104 from FIG. 1A), e.g., when the first and second substrates are aligned in the sandwich configuration. Such alignment may be accomplished manually (e.g., by a user) or automatically (e.g., via an automated alignment mechanism). After or before alignment, spacers may be applied to the first substrate 206 and / or the second substrate 212 to maintain a minimum spacing between the first substrate 206 and the second substrate 212 during sandwiching. In some aspects, the permeabilization solution (e.g., permeabilization solution 305) may be applied to the first substrate 206 and / or the second substrate 212. The first member 204 may then close over the second member 210 and form theny-2785575202412020440 sandwich configuration. Analytes or analyte derivatives (e.g., intermediate agents; e.g., ligation products) may be captured by the capture probes of the array and may be processed for spatial analysis.

[0160] In some embodiments, during the permeabilization step, the image capture device 220 may capture images of the overlap area between the biological sample and the capture probes on the array 106. If more than one first substrates 206 and / or second substrates 212 are present within the sample handling apparatus 200, the image capture device 220 may be configured to capture one or more images of one or more overlap areas.

[0161] Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate. FIGs. 3A-3C depict a side view and a top view of an exemplary angled closure workflow 300 for sandwiching a first substrate (e.g., slide 303) having a biological sample 302 and a second substrate (e.g., slide 304 having capture probes 306) in accordance with some exemplary implementations.

[0162] FIG. 3A depicts the first substrate (e.g., the slide 303 including a biological sample 302) angled over (superior to) the second substrate (e.g., slide 304). As shown, reagent medium (e.g., permeabilization solution) 305 is located on the spacer 310 toward the right-hand side of the side view in FIG. 3A. While FIG. 3A depicts the reagent medium on the right hand side of side view, it should be understood that such depiction is not meant to be limiting as to the location of the reagent medium on the spacer.

[0163] FIG. 3B shows that as the first substrate lowers, and / or as the second substrate rises, the dropped side of the first substrate (e.g., a side of the slide 303 angled toward the second substrate) may contact the reagent medium 305. The dropped side of the first substrate may urge the reagent medium 305 toward the opposite direction (e.g., towards an opposite side of the spacer 310, towards an opposite side of the first substrate relative to the dropped side). For example, in the side view of FIG. 3B the reagent medium 305 may be urged from right to left as the sandwich is formed.

[0164] In some embodiments, the first substrate and / or the second substrate are further moved to achieve an approximately parallel arrangement of the first substrate and the second substrate.

[0165] FIG. 3C depicts a full closure of the sandwich between the first substrate and the second substrate with the spacer 310 contacting both the first substrate and the secondny-2785575202412020440 substrate and maintaining a separation distance and optionally the approximately parallel arrangement between the two substrates. As shown in the top view of FIG. 3C, the spacer 310 fully encloses and surrounds the biological sample 302 and the capture probes 306, and the spacer 310 form the sides of chamber 350 which holds a volume of the reagent medium 305.

[0166] While FIG. 3C depicts the first substrate (e.g., the slide 303 including biological sample 302) angled over (superior to) the second substrate (e.g., slide 304) and the second substrate comprising the spacer 310, it should be understood that an exemplary angled closure workflow can include the second substrate angled over (superior to) the first substrate and the first substrate comprising the spacer 310.

[0167] It may be desirable that the reagent medium be free from air bubbles between the substrates to facilitate transfer of target analytes with spatial information. Additionally, air bubbles present between the substrates may obscure at least a portion of an image capture of a desired region of interest. Accordingly, it may be desirable to ensure or encourage suppression and / or elimination of air bubbles between the two substrates (e.g., slide 303 and slide 304) during a permeabilization step (e.g., step 104). In some aspects, it may be possible to reduce or eliminate bubble formation between the substrates using a variety of filling methods and / or closing methods. In some instances, the first substrate and the second substrate are arranged in an angled sandwich assembly as described herein. For example, during the sandwiching of the two substrates (e.g., the slide 303 and the slide 304), an angled closure workflow may be used to suppress or eliminate bubble formation.

[0168] FIG. 4A is a side view of the angled closure workflow 400 in accordance with some exemplary implementations. FIG. 4B is a top view of the angled closure workflow 400 in accordance with some exemplary implementations. As shown at 405, reagent medium 401 is positioned to the side of the substrate 402 contacting the spring.

[0169] At step 410, the dropped side of the angled substrate 406 contacts the reagent medium 401 first. The contact of the substrate 406 with the reagent medium 401 may form a linear or low curvature flow front that fills uniformly with the slides closed.

[0170] At step 415, the substrate 406 is further lowered toward the substrate 402 (or the substrate 402 is raised up toward the substrate 406) and the dropped side of the substrate 406 may contact and may urge the reagent medium toward the side opposite the dropped side andny-2785575202412020440 creating a linear or low curvature flow front that may prevent or reduce bubble trapping between the substrates.

[0171] At step 420, the reagent medium 401 fills the gap between the substrate 406 and the substrate 402. The linear flow front of the liquid reagent may form by squeezing the 401 volume along the contact side of the substrate 402 and / or the substrate 406. Additionally, capillary flow may also contribute to filling the gap area.

[0172] In some embodiments, the reagent medium (e.g., 105 in FIG 1A) comprises a permeabilization agent. In some embodiments, following initial contact between the biological sample and a permeabilization agent, the permeabilization agent can be removed from contact with the biological sample (e.g., by opening the sample holder). Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross- linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100™, Tween-20™, or sodium dodecyl sulfate (SDS)), and enzymes (e.g., trypsin, proteases (e.g., proteinase K). In some embodiments, the detergent is an anionic detergent (e.g., SDS or N-lauroylsarcosine sodium salt solution).

[0173] In some embodiments, the reagent medium comprises a lysis reagent. Lysis solutions can include ionic surfactants such as, for example, sarkosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents can include, without limitation, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents. In some embodiments, the reagent medium comprises a protease. Exemplary proteases include, e.g., pepsin, trypsin, elastase, and proteinase K. In some embodiments, the reagent medium comprises a nuclease. In some embodiments, the nuclease comprises an RNase. In some embodiments, the RNase is selected from RNase A, RNase C, RNase H, and RNase I. In some embodiments, the reagent medium comprises one or more of sodium dodecyl sulfate (SDS) or a sodium salt thereof, proteinase K, pepsin, N-lauroylsarcosine, and RNase.

[0174] In some embodiments, the reagent medium comprises polyethylene glycol (PEG). In some embodiments, the PEG is from about 2K to about 16K. In some embodiments, the PEG is 2K, 3K, 4K, 5K, 6K, 7K, 8K, 9K, 10K, 11K, 12K, 13K, 14K, 15K, or 16K. In some embodiments, the PEG is present at a concentration from about 2% to 25%, from about 4% to about 23%, from about 6% to about 21%, or from about 8% to about 20% (v / v).ny-2785575202412020440

[0175] In certain embodiments a dried permeabilization reagent is applied or formed as a layer on the first substrate or the second substrate or both prior to contacting the biological sample with the array. For example, a permeabilization reagent can be deposited in solution on the first substrate or the second substrate or both and then dried.

[0176] In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1 minute, about 5 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 18 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 36 minutes, about 45 minutes, or about an hour. In some instances, the aligned portions of the biological sample and the array are in contact with the reagent medium for about 1-60 minutes.

[0177] In some instances, the device is configured to control a temperature of the first and second substrates. In some embodiments, the temperature of the first and second members is lowered to a first temperature that is below room temperature.

[0178] There are at least two methods to associate a spatial barcode with one or more neighboring cells, such that the spatial barcode identifies the one or more cells, and / or contents of the one or more cells, as associated with a particular spatial location. One method is to promote analytes or analyte proxies (e.g., intermediate agents) out of a cell and towards a spatially-barcoded array (e.g., including spatially-barcoded capture probes). Another method is to cleave spatially-barcoded capture probes from an array and promote the spatially-barcoded capture probes towards and / or into or onto the biological sample.

[0179] In some cases, capture probes may be configured to prime, replicate, and consequently yield optionally barcoded extension products from a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent (e.g., a ligation product or an analyte capture agent), or a portion thereof), or derivatives thereof (see, e.g., Section (II)(b)(vii) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663 regarding extended capture probes). In some cases, capture probes may be configured to form ligation products with a template (e.g., a DNA or RNA template, such as an analyte or an intermediate agent, or portion thereof), thereby creating ligation products that serve as proxies for the template.

[0180] As used herein, an “extended capture probe” refers to a capture probe having additional nucleotides added to the terminus (e.g., 3’ or 5’ end) of the capture probe thereby extending the overall length of the capture probe. For example, an “extended 3’ end” indicatesny-2785575202412020440 additional nucleotides were added to the most 3’ nucleotide of the capture probe to extend the length of the capture probe, for example, by polymerization reactions used to extend nucleic acid molecules including templated polymerization catalyzed by a polymerase (e.g., a DNA polymerase or a reverse transcriptase). In some embodiments, extending the capture probe includes adding to a 3’ end of a capture probe a nucleic acid sequence that is complementary to a nucleic acid sequence of an analyte or intermediate agent specifically bound to the capture domain of the capture probe. In some embodiments, the capture probe is extended by a reverse transcriptase. In some embodiments, the capture probe is extended using one or more DNA polymerases. In some embodiments, the extended capture probes include the sequence of the capture domain, the sequence of the spatial barcode of the capture probe, and the complementary sequence of the template used for extension of the capture probe.

[0181] In some embodiments, extended capture probes are amplified (e.g., in bulk solution or on the array) to yield quantities that are sufficient for downstream analysis, e.g., sequencing. In some embodiments, extended capture probes (e.g., DNA molecules) can act as templates for an amplification reaction (e.g., a polymerase chain reaction).

[0182] Additional variants of spatial analysis methods, including in some embodiments, an imaging step, are described in Section (II)(a) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663. Analysis of captured analytes (and / or intermediate agents or portions thereof), for example, including sample removal, extension of capture probes using the capture analyte as a template, sequencing (e.g., of a cleaved extended capture probe and / or a cDNA molecule complementary to an extended capture probe), sequencing on the array (e.g., using, for example, in situ hybridization or in situ ligation approaches), temporal analysis, and / or proximity capture, is described in Section (II)(g) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663. Some quality control measures are described in Section (II)(h) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663.

[0183] Spatial information can provide information of medical importance. For example, the methods described herein can allow for: identification of one or more biomarkers (e.g., diagnostic, prognostic, and / or for determination of efficacy of a treatment) of a disease or disorder; identification of a candidate drug target for treatment of a disease or disorder; identification (e.g., diagnosis) of a subject as having a disease or disorder; identification of stageny-2785575202412020440 and / or prognosis of a disease or disorder in a subject; identification of a subject as having an increased likelihood of developing a disease or disorder; monitoring of progression of a disease or disorder in a subject; determination of efficacy of a treatment of a disease or disorder in a subject; identification of a patient subpopulation for which a treatment is effective for a disease or disorder; modification of a treatment of a subject with a disease or disorder; selection of a subject for participation in a clinical trial; and / or selection of a treatment for a subject with a disease or disorder. Exemplary methods for identifying spatial information of biological and / or medical importance can be found in U.S. Patent Application Publication Nos. 2021 / 0140982, 2021 / 0198741, and 2021 / 0199660, all of which are herein incorporated by reference in their entireties.

[0184] Spatial information can provide information of biological importance. For example, the methods described herein can allow for: identification of transcriptome and / or proteome expression profiles (e.g., in healthy and / or diseased tissue); identification of multiple analyte types in close proximity (e.g., nearest neighbor or proximity based analysis); determination of up- and / or down-regulated genes and / or proteins in diseased tissue; characterization of tumor microenvironments; characterization of tumor immune responses; characterization of cells types and their co-localization in healthy and diseased tissue; and identification of genetic variants within tissues (e.g., based on gene and / or protein expression profiles associated with specific disease or disorder biomarkers).

[0185] Typically, for spatial array-based methods, a substrate functions as a support for direct or indirect attachment of capture probes to features of the array. A “feature” is an entity that acts as a support or repository for various molecular entities used in spatial analysis. In some embodiments, some or all of the features in an array are functionalized for analyte capture. Exemplary substrates are described in Section (II)(c) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663, each of which are herein incorporated by reference in their entireties. Exemplary features and geometric attributes of an array can be found in Sections (II)(d)(i), (II)(d)(iii), and (II)(d)(iv) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663.

[0186] Generally, analytes and / or intermediate agents (or portions thereof) can be captured when contacting a biological sample with a substrate including capture probes (e.g., a substrate with capture probes embedded, spotted, printed, fabricated on the substrate, or any-2785575202412020440 substrate with features (e.g., beads, wells) comprising capture probes). As used herein, “contact,” “contacted,” and / or “contacting,” a biological sample with a substrate refers to any contact (e.g., direct or indirect) such that capture probes can interact (e.g., bind covalently or non-covalently (e.g., hybridize)) with analytes from the biological sample. Capture can be achieved actively (e.g., using electrophoresis) or passively (e.g., using diffusion). Analyte capture is further described in Section (II)(e) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663.

[0187] FIG. 5 is a schematic diagram showing an exemplary capture probe, as described herein. As shown, the capture probe 502 is optionally coupled to a feature 501 by a cleavage domain 503, such as a disulfide linker. The capture probe can include a functional sequence 504 that is useful for subsequent processing. The functional sequence 504 can include all or a part of sequencer specific flow cell attachment sequence (e.g., a P5 or P7 sequence), all or a part of a sequencing primer sequence, (e.g., a R1 primer binding site, a R2 primer binding site), or combinations thereof. The capture probe can also include a spatial barcode 505. The capture probe can also include a unique molecular identifier (UMI) sequence 506. While FIG. 5 shows the spatial barcode 505 as being located upstream (5’) of UMI sequence 506, it is to be understood that capture probes wherein UMI sequence 506 is located upstream (5’) of the spatial barcode 505 is also suitable for use in any of the methods described herein. The capture probe can also include a capture domain 507 to facilitate capture of a target analyte. The capture domain can have a sequence complementary to a sequence of a nucleic acid analyte. The capture domain can have a sequence complementary to a connected probe described herein. The capture domain can have a sequence complementary to an analyte capture sequence present in an analyte capture agent. The capture domain can have a sequence complementary to a splint oligonucleotide. A splint oligonucleotide, in addition to having a sequence complementary to a capture domain of a capture probe, can have a sequence complementary to a sequence of a nucleic acid analyte, a portion of a connected probe described herein, a capture handle sequence described herein, and / or a methylated adaptor described herein.

[0188] FIG. 6 is a schematic illustrating a cleavable capture probe, wherein the cleaved capture probe can enter into a non-permeabilized cell and bind to analytes within the sample. The capture probe 601 contains a cleavage domain 602, a cell penetrating peptide 603, any-2785575202412020440 reporter molecule 604, and a disulfide bond (-S-S-). 605 represents all other parts of a capture probe, for example a spatial barcode and a capture domain.

[0189] FIG. 7 is a schematic diagram of an exemplary multiplexed spatially- barcoded feature. In FIG. 7, the feature 701 can be coupled to spatially-barcoded capture probes, wherein the spatially-barcoded probes of a particular feature can possess the same spatial barcode, but have different capture domains designed to associate the spatial barcode of the feature with more than one target analyte. For example, a feature may include four different types of spatially-barcoded capture probes, each type of spatially-barcoded capture probe possessing the spatial barcode 702. One type of capture probe associated with the feature includes the spatial barcode 702 in combination with a poly(T) capture domain 703, designed to capture mRNA target analytes. A second type of capture probe associated with the feature includes the spatial barcode 702 in combination with a random N-mer capture domain 704 for gDNA analysis. A third type of capture probe associated with the feature includes the spatial barcode 702 in combination with a capture domain complementary to the analyte capture agent of interest 705. A fourth type of capture probe associated with the feature includes the spatial barcode 702 in combination with a capture probe that can specifically bind a nucleic acid molecule 706 that can function in a CRISPR assay (e.g., CRISPR / Cas9). While only four different capture probe-barcoded constructs are shown in FIG. 7, capture-probe barcoded constructs can be tailored for analyses of any given analyte associated with a nucleic acid and capable of binding with such a construct. For example, the schemes shown in FIG. 7 can also be used for concurrent analysis of other analytes disclosed herein, including, but not limited to: (a) mRNA, a lineage tracing construct, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNase-seq, and / or MNase-seq) cell surface or intracellular proteins and metabolites, and a perturbation agent (e.g., a CRISPR crRNA / sgRNA, TALEN, zinc finger nuclease, and / or antisense oligonucleotide as described herein); (c) mRNA, cell surface or intracellular proteins and / or metabolites, a barcoded labeling agent (e.g., the MHC multimers described herein), and a V(D)J sequence of an immune cell receptor (e.g., T-cell receptor). In some embodiments, a perturbation agent can be a small molecule, an antibody, a drug, an aptamer, a miRNA, a physical environmental (e.g., temperature change), or any other known perturbation agents.ny-2785575202412020440

[0190] The functional sequences can generally be selected for compatibility with any of a variety of different sequencing systems, e.g., Ion Torrent Proton or PGM, Illumina sequencing instruments, PacBio, Oxford Nanopore, etc., and the requirements thereof. In some embodiments, functional sequences can be selected for compatibility with non-commercialized sequencing systems. Examples of such sequencing systems and techniques, for which suitable functional sequences can be used, include (but are not limited to) Ion Torrent Proton or PGM sequencing, Illumina sequencing, PacBio SMRT sequencing, and Oxford Nanopore sequencing. Further, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercialized sequencing systems.

[0191] In some embodiments, the spatial barcode 505 and functional sequences 504 are common to all of the probes attached to a given feature. In some embodiments, the UMI sequence 506 of a capture probe attached to a given feature is different from the UMI sequence of a different capture probe attached to the given feature.

[0192] FIG. 8 depicts an exemplary arrangement of barcoded features within an array. From left to right, FIG. 8 shows (L) a slide including six spatially-barcoded arrays, (C) an enlarged schematic of one of the six spatially-barcoded arrays, showing a grid of barcoded features in relation to a biological sample, and (R) an enlarged schematic of one section of an array, showing the specific identification of multiple features within the array (labelled as ID578, ID579, ID560, etc.).

[0193] In some embodiments, more than one analyte type (e.g., nucleic acids and proteins) from a biological sample can be detected (e.g., simultaneously or sequentially) using any appropriate multiplexing technique, such as those described in Section (IV) of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663.

[0194] In some cases, spatial analysis can be performed by attaching and / or introducing a molecule (e.g., a peptide, a lipid, or a nucleic acid molecule) having a barcode (e.g., a spatial barcode) to a biological sample (e.g., to a cell in a biological sample). In some embodiments, a plurality of molecules (e.g., a plurality of nucleic acid molecules) having a plurality of barcodes (e.g., a plurality of spatial barcodes) are introduced to a biological sample (e.g., to a plurality of cells in a biological sample) for use in spatial analysis. In some embodiments, after attaching and / or introducing a molecule having a barcode to a biological sample, the biological sample can be physically separated (e.g., dissociated) into single cells orny-2785575202412020440 cell groups for analysis. Some such methods of spatial analysis are described in Section (III) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663.

[0195] In some cases, spatial analysis can be performed by detecting multiple oligonucleotides that hybridize to an analyte. In some instances, for example, spatial analysis can be performed using RNA-templated ligation (RTL). Methods of RTL have been described previously. See, e.g., Credle et al., Nucleic Acids Res. 2017 Aug 21; 45(14):e128. Typically, RTL includes hybridization of two oligonucleotides to adjacent sequences on an analyte (e.g., an RNA molecule, such as an mRNA molecule). In some instances, the oligonucleotides are DNA molecules. In some instances, one of the oligonucleotides includes at least two ribonucleic acid bases at the 3’ end and / or the other oligonucleotide includes a phosphorylated nucleotide at the 5’ end. In some instances, one of the two oligonucleotides includes a capture domain (e.g., a poly(A) sequence, a non-homopolymeric sequence). After hybridization to the analyte, a ligase (e.g., a T4 RNA ligase (Rnl2), a PBCV-1 DNA Ligase or Chlorella virus DNA Ligase, a single- stranded DNA ligase, or a T4 DNA ligase) ligates the two oligonucleotides together, creating a ligation product. In some instances, the two oligonucleotides hybridize to sequences that are not adjacent to one another. For example, hybridization of the two oligonucleotides creates a gap between the hybridized oligonucleotides. In some instances, a polymerase (e.g., a DNA polymerase) can extend one of the oligonucleotides prior to ligation. After ligation, the ligation product is released from the analyte. In some instances, the ligation product is released using an endonuclease (e.g., RNase H). In some instances, RNase H digests the RNA from a RNA:DNA hybrid, and in cases where the RNA strand is tethered (e.g., to a hydrogel matrix), RNase can be used to release the DNA strand. In some instances, the ligation product is removed using heat. In some instances, the ligation product is removed using KOH. The released ligation product can then be captured by capture probes (e.g., instead of direct capture of an analyte) on an array, optionally amplified, and sequenced, thus determining the location and optionally the abundance of the analyte in the biological sample.

[0196] In some instances, one or both of the oligonucleotides may hybridize to genomic DNA (gDNA) which can lead to false positive sequencing data from ligation events on gDNA (off target) in addition to the desired (on target) ligation events on target nucleic acids, (e.g., mRNA). Thus, in some embodiments, the disclosed methods can include contacting the biological sample with a deoxyribonuclease (DNase). The DNase can be an endonuclease orny-2785575202412020440 exonuclease. In some embodiments, the DNase digests single- and / or double-stranded DNA. Suitable DNases include, without limitation, a DNase I and a DNase II. Use of a DNase as described can mitigate false positive sequencing data from off target gDNA ligation events.

[0197] A non-limiting example of templated ligation methods disclosed herein is depicted in FIG. 9A. After a biological sample is contacted with a substrate including a plurality of capture probes and contacted with (a) a first probe 901 having a target-hybridization sequence 903 and a primer sequence 902 and (b) a second probe 904 having a target-hybridization sequence 905 and a capture domain (e.g., a poly-A sequence) 906, the first probe 901 and a second probe 904 hybridize 910 to an analyte 907. A ligase 921 ligates 920 the first probe to the second probe thereby generating a ligation product 922. The ligation product is released 930 from the analyte 931 by digesting the analyte using an endoribonuclease 932. The sample is permeabilized 940 and the ligation product 941 is able to hybridize to a capture probe on the substrate. Methods and composition for spatial detection using templated ligation have been described in PCT Publ. No. WO 2021 / 133849 A1, U.S. Pat. Nos. 11,332,790 and 11,505,828, all of which are herein incorporated by reference in its entireties.

[0198] In some embodiments, as shown in FIG. 9B, the ligation product 9001 includes a capture probe capture domain 9002, which can bind to a capture probe 9003 (e.g., a capture probe immobilized, directly or indirectly, on a substrate 9004). In some embodiments, methods provided herein include contacting 9005 a biological sample with a substrate 9004, wherein the capture probe 9003 is affixed to the substrate (e.g., immobilized to the substrate, directly or indirectly). In some embodiments, the capture probe capture domain 9002 of the ligated product specifically binds to the capture domain 9006. The capture probe can also include a unique molecular identifier (UMI) 9007, a spatial barcode 9008, a functional sequence 9009, and a cleavage domain 9010.

[0199] In some embodiments, methods provided herein include permeabilization of the biological sample such that the capture probe can more easily capture the ligation products (i.e., as compared to no permeabilization). In some embodiments, reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with the RT reagents can extend the capture probes 9011 to produce spatially-barcoded full-length cDNA 9012 and 9013 from the captured ligation products (e.g., ligation products).ny-2785575202412020440

[0200] In some embodiments, the extended ligation products can be denatured 9014 from the capture probe and transferred (e.g., to a clean tube) for amplification, and / or library construction. The spatially-barcoded ligation products can be amplified 9015 via PCR prior to library construction. P59016, i59017, i79018, and P79019, and can be used as sequencing probes (P5 and P7) and sample indexes (i5 and i7) when utilizing Illumina sequencing platforms. The amplicons can then be sequenced using paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites.

[0201] In some embodiments, detection of one or more analytes (e.g., protein analytes) comprises using one or more analyte capture agents. As used herein, an “analyte capture agent” refers to an agent that interacts with an analyte (e.g., an analyte in a biological sample) and with a capture probe (e.g., a capture probe attached to a substrate or a feature) to identify the analyte. In some embodiments, the analyte capture agent comprises: (i) an analyte binding moiety (e.g., an moiety that directly or indirectly binds to an analyte), for example, an antibody or antigen-binding fragment thereof; (ii) an analyte binding moiety barcode, e.g., a nucleic acid barcode; and (iii) an analyte capture sequence. In some embodiments, the analyte binding moiety is an antibody. In some embodiments, the analyte binding moiety is an antibody that binds to a target protein in a biological sample. As used herein, the term “analyte binding moiety barcode” refers to a barcode that is associated with, corresponds to, and / or identifies the analyte binding moiety. As used herein, the term “analyte capture sequence” refers to a region or moiety configured to hybridize to, bind to, couple to, or otherwise interact with a capture domain of a capture probe. In some cases, an analyte binding moiety barcode (or portion thereof) may be able to be removed (e.g., cleaved) from the analyte capture agent. Additional description of analyte capture agents can be found in Section (II)(b)(ix) of U.S. Patent Application Publication No. 2022 / 0010367 and / or Section (II)(b)(viii) U.S. Patent Application Publication No. 2020 / 0277663, each of which are herein incorporated by reference in their entireties.

[0202] FIG. 10 is a schematic diagram of an exemplary analyte capture agent 1002 comprised of an analyte-binding moiety 1004 and an analyte associated oligonucleotide 1008 which comprises a binding moiety barcode domain and an analyte capture sequence. The exemplary analyte-binding moiety 1004 is a molecule capable of binding to an analyte 1006 and the analyte capture agent is capable of interacting with a spatially-barcoded capture probe. The analyte-binding moiety can bind to the analyte 1006 with high affinity and / or with highny-2785575202412020440 specificity. The analyte capture agent can include an analyte associated oliognucleoide 1008 which comprises an analyte-binding moiety barcode domain which serves to identify the analyte binding moiety, and a capture sequence which can hybridize to at least a portion or an entirety of a capture domain of a capture probe. The analyte-binding moiety 1004 can include a polypeptide and / or an aptamer. The analyte-binding moiety 1004 can include an antibody or antibody fragment (e.g., an antigen-binding fragment).

[0203] FIG. 11 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1124 and an analyte capture agent 1126. The feature- immobilized capture probe 1124 can include a spatial barcode 1108 as well as functional sequences 1106 and a UMI 1110, as described elsewhere herein. The capture probe can be affixed 1104 to a feature such as a bead 1102. The capture probe can also include a capture domain 1112 that is capable of binding to an analyte capture agent 1126. The analyte-binding moiety barcode domain of the analyte capture agent 1126 can include a functional sequence 1118, analyte binding moiety barcode 1116, and an analyte capture sequence 1114 that is capable of binding (e,g, hybridizing) to the capture domain 1112 of the capture probe 1124. The analyte capture agent can also include a linker 1120 that allows the analyte-binding moiety barcode domain (e.g., including the functional sequence 1118, analyte binding barcode 1116, and analyte capture sequence 1114) to couple to the analyte binding moiety 1122. In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a photo-cleavable linker, a UV-cleavable linker, or an enzyme cleavable linker. In some instances, the cleavable linker is a disulfide linker. A disulfide linker can be cleaved by use of a reducing agent, such as dithiothreitol (DTT), Beta-mercaptoethanol (BME), or Tris (2-carboxyethyl) phosphine (TCEP).

[0204] During analysis of spatial information, sequence information for a spatial barcode associated with an analyte is obtained, and the sequence information can be used to provide information about the spatial distribution of the analyte in the biological sample. Various methods can be used to obtain the spatial information. In some embodiments, specific capture probes and the analytes they capture are associated with specific locations in an array of features on a substrate. For example, specific spatial barcodes can be associated with specific array locations prior to array fabrication, and the sequences of the spatial barcodes can be stored (e.g., in a database) along with specific array location information, so that each spatial barcode uniquely maps to a particular array location.ny-2785575202412020440

[0205] Alternatively, specific spatial barcodes can be deposited at predetermined locations in an array of features during fabrication such that at each location, only one type of spatial barcode is present so that spatial barcodes are uniquely associated with a single feature of the array. Where necessary, the arrays can be decoded using any of the methods described herein so that spatial barcodes are uniquely associated with array feature locations, and this mapping can be stored as described above.

[0206] When sequence information is obtained for capture probes and / or analytes during analysis of spatial information, the locations of the capture probes and / or analytes can be determined by referring to the stored information that uniquely associates each spatial barcode with an array feature location. In this manner, specific capture probes and captured analytes are associated with specific locations in the array of features. Each array feature location represents a position relative to a coordinate reference point (e.g., an array location, a fiducial marker) for the array. Accordingly, each feature location has an “address” or location in the coordinate space of the array.

[0207] Some exemplary spatial analysis workflows are described in the Exemplary Embodiments section of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663. See, for example, the Exemplary embodiment starting with “In some non- limiting examples of the workflows described herein, the sample can be immersed…” of PCT Publication No. WO2020 / 176788 and / or U.S. Patent Application Publication No. 2020 / 0277663. See also, e.g., the Visium Spatial Gene Expression Reagent Kits User Guide (e.g., Rev F, dated January 2022); and / or the Visium Spatial Gene Expression Reagent Kits - Tissue Optimization User Guide (e.g., Rev E, dated February 2022).

[0208] In some embodiments, spatial analysis can be performed using dedicated hardware and / or software, such as any of the systems described in Sections (II)(e)(ii) and / or (V) of US Patent Application Publication Nos. 2022 / 0010367 and / or 2020 / 0277663, or any of one or more of the devices or methods described in Sections Control Slide for Imaging, Methods of Using Control Slides and Substrates for, Systems of Using Control Slides and Substrates for Imaging, and / or Sample and Array Alignment Devices and Methods, Informational labels of PCT Publication No. WO2020 / 123320.

[0209] Suitable systems for performing spatial analysis can include components such as a chamber (e.g., a flow cell or sealable, fluid-tight chamber) for containing a biologicalny-2785575202412020440 sample. The biological sample can be mounted for example, in a biological sample holder. One or more fluid chambers can be connected to the chamber and / or the sample holder via fluid conduits, and fluids can be delivered into the chamber and / or sample holder via fluidic pumps, vacuum sources, or other devices coupled to the fluid conduits that create a pressure gradient to drive fluid flow. One or more valves can also be connected to fluid conduits to regulate the flow of reagents from reservoirs to the chamber and / or sample holder.

[0210] The systems can optionally include a control unit that includes one or more electronic processors, an input interface, an output interface (such as a display), and a storage unit (e.g., a solid state storage medium such as, but not limited to, a magnetic, optical, or other solid state, persistent, writeable and / or re-writeable storage medium). The control unit can optionally be connected to one or more remote devices via a network. The control unit (and components thereof) can generally perform any of the steps and functions described herein. Where the system is connected to a remote device, the remote device (or devices) can perform any of the steps or features described herein. The systems can optionally include one or more detectors (e.g., CCD, CMOS) used to capture images. The systems can also optionally include one or more light sources (e.g., LED-based, diode-based, lasers) for illuminating a sample, a substrate with features, analytes from a biological sample captured on a substrate, and various control and calibration media.

[0211] The systems can optionally include software instructions encoded and / or implemented in one or more of tangible storage media and hardware components such as application specific integrated circuits. The software instructions, when executed by a control unit (and in particular, an electronic processor) or an integrated circuit, can cause the control unit, integrated circuit, or other component executing the software instructions to perform any of the method steps or functions described herein.

[0212] In some cases, the systems described herein can detect (e.g., register an image) the biological sample on the array. Exemplary methods to detect the biological sample on an array are described in PCT Publication No. WO2021 / 102003 and / or U.S. Patent Application Publication No. 2021 / 0150707, each of which is incorporated herein by reference in their entireties.

[0213] Prior to transferring analytes from the biological sample to the array of features on the substrate, the biological sample can be aligned with the array. Alignment of any-2785575202412020440 biological sample and an array of features including capture probes can facilitate spatial analysis, which can be used to detect differences in analyte presence and / or level within different positions in the biological sample, for example, to generate a three-dimensional map of the analyte presence and / or level. Exemplary methods to generate a two- and / or three-dimensional map of the analyte presence and / or level are described in PCT Publication No. WO2020 / 053655 and / or US Patent Application Publication No. 2022 / 0062246, and spatial analysis methods are generally described in PCT Publication No. WO2021 / 102039 and / or U.S. Patent Application Publication No. 2021 / 0155982, each of which is incorporated herein by reference in their entireties.

[0214] In some cases, a map of analyte presence and / or level can be aligned to an image of a biological sample using one or more fiducial markers, e.g., objects placed in the field of view of an imaging system which appear in the image produced, as described in the Substrate Attributes Section, Control Slide for Imaging Section of PCT Publication Nos. WO2020 / 123320, WO 2021 / 102005, and / or U.S. Patent Application Publication No. 2021 / 0158522, each of which is incorporated herein by reference in their entireties. Fiducial markers can be used as a point of reference or measurement scale for alignment (e.g., to align a sample and an array, to align two substrates, to determine a location of a sample or array on a substrate relative to a fiducial marker) and / or for quantitative measurements of sizes and / or distances. II. MATRIX-ASSISTED SPATIAL ANALYSIS

[0215] In some embodiments, array-based spatial analysis methods disclosed herein involve tethering nucleic acid molecules to a matrix in which a biological sample is embedded. The tethered nucleic acid molecules can be present in the biological sample (such as cellular DNA or RNA), or applied to the biological sample (such as a nucleic acid probe targeting a cellular DNA or RNA), or generated in the biological sample as a proxy of a cellular DNA or RNA. In some embodiments, a method disclosed herein further comprises removing at least a subset of the proteins, lipids, or other cellular components in the biological sample from the matrix, thereby leaving the nucleic acid molecules tethered to the matrix. In some embodiments, the tethered nucleic acid molecules can be transferred to a spatial array on a substrate, wherein the spatial array comprises a plurality of features and wherein each feature is associated with a unique spatial location on the array. Subsequent analysis of transferred nucleic acid molecules to the spatial array can include determining the identities of the transferred nucleic acid molecules (and the identities of the corresponding analytes) and the spatial location of the transferredny-2785575202412020440 nucleic acid molecules (and the spatial locations of the corresponding analytes) within the original biological sample. Since the array and the biological sample can be aligned (e.g., such that a location in the biogical sample corresponds to the location of a feature within the array), the spatial location of an analyte within the biological sample can be determined based on the feature to which the corresponding nucleic acid molecule is bound (e.g., directly or indirectly) on the array, and the feature’s relative spatial location within the array, which is determined by the spatial barcodes on the capture probes at each feature to which the transferred nucleic acid molecules are hybridized or ligated.

[0216] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: embedding the biological sample in a matrix and tethering a target nucleic acid in the biological sample to the matrix; clearing at least a subset of proteins, lipids, or other cellular components in the biological sample from the matrix; releasing the target nucleic acid from the matrix; capturing (e.g., via nucleic acid hybridization and / or ligation) the released target nucleic acid to a capture domain of a capture probe on an array (e.g., at a feature), wherein the capture probe further comprises a spatial barcode; and determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the target nucleic acid comprises one or more of DNA or RNA. In some embodiments, the target nucleic acid is RNA such as mRNA. In some embodiments, the target nucleic acid comprises DNA and / or RNA. In some embodiments, the target nucleic acid comprises one or more different DNA sequences and / or one or more different RNA sequences. In some embodiments, the target nucleic acid comprises one or more different DNA molecules and / or one or more different RNA molecules.

[0217] In some embodiments, the matrix is a hydrogel matrix. In some embodiments, the tethering comprises tethering a 3’ end of the target nucleic acid to the matrix. In some embodiments, the tethering comprises tethering a 5’ end of the target nucleic acid to the matrix. In some embodiments, the tethering comprises enzymatic tethering. In some embodiments, the tethering comprises non-enzymatic tethering. In some embodiments, the tethering comprises covalent attachment. In some embodiments, the tethering comprises non- covalent attachment. In some embodiments, the tethering comprises use of an attachment agent. In some embodiments, the tethering comprises a periodate oxidation reaction or a reaction with boronic acid.ny-2785575202412020440

[0218] In some embodiments, the target nucleic acid is released via cleaving a linker between the matrix (e.g., hydrogel) and the target nucleic acid (e.g., the matrix is not dissolved) and / or via cleaving the crosslinker(s) within the matrix (e.g., dissolving the matrix). In cases where the matrix can be dissolved, any crosslinker having one or more disulfide bonds can be used. In some embodiments, the tethering comprises tethering the target nucleic acid to the matrix (e.g., hydrogel) via a linker. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the cleavable linker comprises a disulfide bond, a diol, and / or a serinol. In some embodiments, the matrix is formed using N,N′-Bis(acryloyl)cystamine (BAC) as a crosslinker. In some embodiments, the linker is a photocleavable linker.

[0219] In some embodiments, the linker tethering the target nucleic acid to the matrix (e.g., hydrogel) can be cleavable without cleaving or dissolving the matrix. In some embodiments, the matrix (e.g., hydrogel) can be cleavable or dissolvable, and the linker itself may but does not need to be cleavable. In some embodiments, a crosslinker linker forming the matrix (e.g., hydrogel) can be cleavable, for example, a crosslinker comprising a disulfide bond (e.g., N,N′-Bis(acryloyl)cystamine) can be used to form the matrix embedding the biological sample. In some embodiments, a crosslinker comprising a disulfide bond (e.g., N,N′- Bis(acryloyl)cystamine) can be used as a reversible crosslinker for a polyacrylamide gel, and the disulfide bond can be broken with a reducing agent.

[0220] In some embodiments, a crosslinker comprising a cleavable bond (e.g., disulfide bond) can be used in cases where it is desirable to release tethered targets by dissolving a matrix formed using the crosslinker through contact with a chemical agent configured to cleave the cleavable bond. In some embodiments, a non-dissolvable or dissolvable matrix (e.g., hydrogel) can be used to tethered target nucleic acids. In some embodiments, a non-cleavable or cleavable linker connecting the target nucleic acid to the matrix can be used. In some embodiments, a polymeric matrix composition can be used.

[0221] In some embodiments, the target-tethering approach can comprise a dissolvable / reversible hydrogel and / or cleavable linker between the target and the matrix. Examples include and are not limited to thermo-responsive crosslinkers / matrix, e.g., N- Isopropylacrylamide (NIPAm) and derivatives / compounds; photo-responsive crosslinkers, e.g. azobenzene-based molecules or spiropyran; crosslinkers based on dynamic covalent bonds, like disulfides (e.g., N,N′-Bis(acryloyl)cystamine), boronate esters diols (e.g., N,N′-(1,2-Dihydroxyny-2785575202412020440 Ethylene)bis-acrylamide) or imines; pH responsive crosslinkers; DNA or peptide-based hydrogels – temperature, pH, enzymatic activity dependent; and a combination of any one or more of the chemistries. Additional examples of linkage that can be used in the present disclosure can include and are not limited to:

[0222] In some embodiments, the method comprises removing at least a subset of ribosomes from the biological sample or from the matrix. In some embodiments, the releasing comprises contacting the matrix with a chemical agent configured to cleave a cleavable linker between the target nucleic acid and the matrix. In some embodiments, the releasing comprises contacting the matrix with a chemical agent configured to cleave the crosslinker(s) within the matrix (e.g., dissolving the matrix). In some embodiments, the releasing comprises illuminating the matrix with light, thereby cleaving a photocleavable linker between the target nucleic acid and the matrix. In some embodiments, the releasing comprises altering the pH of a solution in contact with the matrix, thereby releasing the tethered target nucleic acid. In some embodiments,ny-2785575202412020440 a restriction site is engineered at a point of attachment to the matrix. In some embodiments, a restriction site in a linker can be cleaved to release the target nucleic acid from the matrix.

[0223] In some embodiments, the target nucleic acid is tethered to the matrix via a boronate ester bond. In some embodiments, the boronate ester bond is formed between a boronic acid moiety and 3’ diols of the target nucleic acid which is an RNA. In some embodiments, the matrix comprises a boronic acid-based hydrogel matrix. In some embodiments, the releasing comprises exposing the matrix to a temperature of about 40°C to about 75°C or higher, thereby releasing the tethered target nucleic acid. In some embodiments, the releasing comprises exposing the matrix to a temperature of about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, or about 75°C, thereby releasing the tethered target nucleic acid. In some embodiments, the releasing comprises exposing the matrix to a temperature of about 60°C or lower (e.g., between 40°C and 60°C), thereby releasing the tethered target nucleic acid. In some embodiments, since tissue stability is less of a concern once target nucleic acids are tethered to the matrix (e.g., hydrogel), the releasing can comprise exposing the matrix to a temperature of higher than 60°C. In some embodiments, the matrix is a hydrogel matrix comprising poly(N-isopropylacrylamide).

[0224] In some embodiments, the target nucleic acid comprises a cellular nucleic acid in the biological sample. In some embodiments, the target nucleic acid comprises an endogenous nucleic acid of a cell in the biological sample, a product of the cellular nucleic acid generated in situ in the biological sample, a nucleic acid probe that directly or indirectly binds to the cellular nucleic acid, or a product of the nucleic acid probe generated in situ in the biological sample.

[0225] In some embodiments, the matrix and the biological sample embedded therein are on a first solid support (e.g., a sample substrate), and the spatial array is on a second solid support (e.g., an array substrate). In some embodiments, the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, is determined using nucleic acid sequencing. In some embodiments, the nucleic acid sequencing comprises sequencing by synthesis, sequencing by ligation, sequencing by binding, sequencing by avidity, sequencing by hybridization, or any combination thereof.

[0226] In some embodiments, the method further comprises directing the released target nucleic acid to the capture probe. In some embodiments, the directing comprises directingny-2785575202412020440 via a capacitor, thereby generating an electrophoretic migration of the released target nucleic acid from the matrix to the spatial array. In some embodiments, at least one plate of the capacitor comprises a coating of indium tin oxide (ITO).

[0227] In some embodiments, the matrix is a first hydrogel matrix and the capture probe is affixed on the array and embedded in a second hydrogel matrix. In some embodiments, the first hydrogel matrix and the second hydrogel matrix are of the same composition. In some embodiments, the first hydrogel matrix and the second hydrogel matrix are of different compositions.

[0228] In some embodiments, the method comprises determining a spatial expression profile of the target nucleic acid in the biological sample. In some embodiments, a plurality of different target nucleic acids are tethered to the matrix, and after partial or complete sample removal (e.g., by clearing), the tethered target nucleic acids are released from the matrix and hybridized or ligated to capture domains of capture probes on the array. In some embodiments, the method comprises removing the biological sample from the matrix. In some embodiments, removing the biological sample comprises clearing the biological sample. In some embodiments, the method comprises removing a subset of cellular components from the biological sample in the matrix. In some embodiments, removing the subset of cellular components from the biological sample comprises clearing the biological sample. In some embodiments, the clearing comprises digesting and removing the embedded biological sample from the matrix.

[0229] In some embodiments, the biological sample is a fresh tissue sample, a frozen tissue sample, or a fixed tissue sample. In some embodiments, the biological sample is a fresh frozen (FF) tissue section or a formalin-fixed paraffin-embedded (FFPE) tissue section. In some embodiments, the method comprises fixing the biological sample prior to embedding in the matrix, and decrosslinking the embedded biological sample prior to or during the clearing.

[0230] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: contacting a biological sample comprising a target nucleic acid with an attachment agent comprising a boronic acid moiety capable of covalently reacting with at least one 2’,3’ vicinal diol of the target nucleic acid and an attachment moiety capable of attaching covalently or noncovalently to a matrix-forming agent in the biological sample, wherein the biological sample and the attachment agent are contacted under conditions suitableny-2785575202412020440 to form a covalent bond between the boronic acid moiety and the 2’,3’ vicinal diol of the target nucleic acid; contacting the biological sample with a matrix-forming agent, thereby forming a matrix embedding the biological sample and tethering the target nucleic acid to the matrix; clearing the embedded biological sample from the matrix; releasing the target nucleic acid from the matrix; capturing the target nucleic acid by a capture probe on an array, wherein the capture probe comprises a capture domain and a spatial barcode; and determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0231] In some embodiments, disclosed herein is a method for analyzing a biological sample, the method comprising: contacting the biological sample comprising a fragmented target nucleic acid with a 3’ phosphatase to provide a fragmented target nucleic acid comprising a 2’,3’-vicinal diol moiety; contacting the biological sample with a formylation reagent, wherein the formylation reagent converts the 2’,3’-vicinal diol moiety into a 2’,3’-dialdehyde moiety, optionally wherein the formylation reagent comprises sodium (meta)periodate; contacting the biological sample with an attachment agent and a matrix-forming agent, the attachment agent comprising at least one aldehyde-reactive group capable of forming a covalent bond with at least one aldehyde of the 2’,3’-dialdehyde moiety of the fragmented target nucleic acid and an attachment moiety capable of attaching covalently or noncovalently to the matrix-forming agent, thereby forming a matrix embedding the biological sample and tethering the fragmented target nucleic acid to the matrix; clearing the embedded biological sample from the matrix; releasing the fragmented target nucleic acid from the matrix; hybridizing the fragmented target nucleic acid to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode; and determining the sequence of all or a portion of the fragmented target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0232] In some embodiments, the capture probe further comprises a cleavage domain, a functional domain, a unique molecular identifier, or a combination thereof. In some embodiments, the cleavage domain comprises a cleavage sequence. In some embodiments, the functional domain comprises a functional sequence such as a primer binding sequence or a complement thereof.ny-2785575202412020440 A. Sample Preparation and Processing

[0233] In some embodiments, a biological sample and / or an array disclosed herein is provided on a substrate. A substrate herein can be any support that is insoluble in aqueous liquid and which allows for positioning of biological samples, analytes, features, and / or reagents (e.g., probes such as capture probes each comprising a capture domain and a spatial barcode) on the support. In some embodiments, a biological sample can be attached to a substrate. Attachment of the biological sample can be irreversible or reversible, depending upon the nature of the sample and subsequent steps in the analytical method. In certain embodiments, the sample can be attached to the substrate reversibly by applying a suitable polymer coating to the substrate, and contacting the sample to the polymer coating. The sample can then be detached from the substrate, e.g., using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers that are suitable for this purpose. In some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate attachment of the sample to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, poly-lysine, antibodies, and polysaccharides.

[0234] A variety of steps can be performed to prepare or process a biological sample for and / or during an assay. Except where indicated otherwise, the preparative or processing steps described below can generally be combined in any manner and in any order to appropriately prepare or process a particular sample for and / or analysis. (i) Sample Preparation

[0235] A biological sample can be harvested from a subject (e.g., via surgical biopsy, whole subject sectioning) or grown in vitro on a growth substrate or culture dish as a population of cells, and prepared for analysis as a tissue slice or tissue section. Grown samples may be sufficiently thin for analysis without further processing steps. Alternatively, grown samples, and samples obtained via biopsy or sectioning, can be prepared as thin tissue sections using a mechanical cutting apparatus such as a vibrating blade microtome. As another alternative, in some embodiments, a thin tissue section can be prepared by applying a touch imprint of a biological sample to a suitable substrate material.

[0236] The thickness of the tissue section can be a fraction of (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1) the maximum cross-sectional dimension of a cell. However, tissue sections having a thickness that is larger than the maximum cross-section cell dimensionny-2785575202412020440 can also be used. For example, cryostat sections can be used, which can be, e.g., 10-20 µm thick. More generally, the thickness of a tissue section typically depends on the method used to prepare the section and the physical characteristics of the tissue, and therefore sections having a wide variety of different thicknesses can be prepared and used. For example, the thickness of the tissue section can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40, or 50 µm. Thicker sections can also be used if desired or convenient, e.g., at least 70, 80, 90, or 100 µm or more. Typically, the thickness of a tissue section is between 1-100 µm, 1-50 µm, 1-30 µm, 1-25 µm, 1-20 µm, 1-15 µm, 1-10 µm, 2-8 µm, 3-7 µm, or 4-6 µm, but as mentioned above, sections with thicknesses larger or smaller than these ranges can also be analyzed. In some embodiments, the thickness of a tissue section is about 5 µm, about 10 µm, or about 15 µm. In some embodiments, a tissue section for analysis using a method disclosed herein is a fresh frozen tissue section and is about 10 µm thick. In some embodiments, a tissue section for analysis using a method disclosed herein is a fixed tissue section (e.g., a formalin- fixation and paraffin-embedded tissue section) and is about 5 µm thick.

[0237] Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy sample by performing serial sectioning of the biopsy sample using a sectioning blade. Spatial information among the serial sections can be preserved in this manner, and the sections can be analyzed successively to obtain three-dimensional information about the biological sample.

[0238] In some embodiments, the biological sample (e.g., a tissue section as described above) can be prepared by deep freezing at a temperature suitable to maintain or preserve the integrity (e.g., the physical characteristics) of the tissue structure. The frozen tissue sample can be sectioned, e.g., thinly sliced, onto a substrate surface using any number of suitable methods. For example, a tissue sample can be prepared using a chilled microtome (e.g., a cryostat) set at a temperature suitable to maintain both the structural integrity of the tissue sample and the chemical properties of the nucleic acids in the sample. Such a temperature can be, e.g., less than -15°C, less than -20°C, or less than -25°C.

[0239] In some embodiments, the biological sample can be prepared using formalin- fixation and paraffin-embedding (FFPE), which are established methods. In some embodiments, cell suspensions and other non-tissue samples can be prepared using formalin-fixation and paraffin-embedding. Following fixation of the sample and embedding in a paraffin or resinny-2785575202412020440 block, the sample can be sectioned as described above. Prior to analysis, the paraffin-embedding material can be removed from the tissue section (e.g., deparaffinization) by incubating the tissue section in an appropriate solvent (e.g., xylene) followed by a rinse (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes).

[0240] As an alternative to formalin fixation described above, a biological sample can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, a sample can be fixed via immersion in ethanol, methanol, acetone, paraformaldehyde (PFA)-Triton, and combinations thereof.

[0241] In some embodiments, the methods provided herein comprises one or more post-fixing (also referred to as postfixation) steps. In some embodiments, one or more post- fixing step is performed after contacting a sample with a polynucleotide disclosed herein, e.g., one or more probes such as a probe or probe set configured to be ligated using RNA as a template. In some embodiments, one or more post-fixing step is performed after a hybridization complex comprising a probe and a target is formed in a sample. In some embodiments, one or more post-fixing step is performed prior to a ligation reaction disclosed herein.

[0242] In some embodiments, a method disclosed herein comprises de-crosslinking a fixed or crosslinked sample, e.g., a reversibly crosslinked biological sample. The de- crosslinking does not need to be complete. In some embodiments, only a portion of crosslinked molecules in the reversibly cross-linked biological sample are de-crosslinked and allowed to migrate.

[0243] In some embodiments, a biological sample can be permeabilized to facilitate migration of species (such as analytes from within the biological sample) to the capture probes on the array. If a sample is not permeabilized sufficiently, the migration of species from the sample may be too low to enable adequate analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample can be lost. Hence, a balance between permeabilizing the tissue sample enough to obtain good signal intensity while still maintaining the spatial resolution of the analyte distribution in the sample is desirable.

[0244] In general, a biological sample can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g.,ny-2785575202412020440 paraformaldehyde), detergents (e.g., saponin, Triton X-100™ or Tween-20™), and enzymes (e.g., trypsin, proteases). In some embodiments, the biological sample can be incubated with a cellular permeabilizing agent to facilitate permeabilization of the sample. Additional methods for sample permeabilization are described, for example, in Jamur et al., Method Mol. Biol. 588:63- 66, 2010, the entire contents of which are incorporated herein by reference. Any suitable method for sample permeabilization can generally be used in connection with the samples described herein.

[0245] Additional reagents can be added to a biological sample to perform various functions prior to analysis of the sample. In some embodiments, DNase and RNase inactivating agents (e.g., proteinase K) or inhibitors such as RNase inhibitors, and / or chelating agents such as EDTA, can be added to the sample. For example, a method disclosed herein may comprise a step for increasing accessibility of a nucleic acid for binding, e.g., a denaturation step to open up DNA in a cell for hybridization by a probe. (ii) Sample Embedding in Matrix

[0246] In some embodiments, the biological sample can be embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material, and activating the polymer material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample. Biological samples can include analytes (e.g., protein, RNA, and / or DNA) embedded in a 3D matrix. In some embodiments, amplicons (e.g., rolling circle amplification products) derived from or associated with analytes (e.g., protein, RNA, and / or DNA) can be embedded in a 3D matrix. In some embodiments, a 3D matrix may comprise a network of natural molecules and / or synthetic molecules that are chemically and / or enzymatically linked, e.g., by crosslinking. In some embodiments, a 3D matrix may comprise a synthetic polymer. In some embodiments, a 3D matrix comprises a hydrogel.

[0247] In some aspects, a biological sample can be embedded in any of a variety of other embedding materials to provide structural substrate to the sample prior to sectioning and other handling steps. In some cases, the embedding material can be removed e.g., prior tony-2785575202412020440 analysis of tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylate resins), epoxies, and agar.

[0248] In some embodiments, the biological sample can be embedded in a matrix (e.g., a hydrogel matrix). Embedding the sample in this manner typically involves contacting the biological sample with a hydrogel such that the biological sample becomes surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material, and activating the polymer material to form a hydrogel. In some embodiments, the hydrogel is formed such that the hydrogel is internalized within the biological sample.

[0249] In some embodiments, the biological sample is immobilized in the hydrogel via cross-linking of the polymer material that forms the hydrogel. Cross-linking can be performed chemically and / or photochemically, or alternatively by any other suitable hydrogel- formation method.

[0250] In some embodiments, the biological sample is reversibly cross-linked prior to or during an assay disclosed herein. In some aspects, the analytes, polynucleotides and / or amplification product (e.g., amplicon) of an analyte or a probe bound thereto can be anchored to a polymer matrix. For example, the polymer matrix can be a hydrogel. In some embodiments, one or more of the target analytes, analyte proxies and / or amplification products (e.g., amplicons) thereof can be modified to contain functional groups that can be used as an anchoring site to attach the target analytes, analyte proxies and / or amplification products to a polymer matrix. In some embodiments, a modified probe comprising oligo dT may be used to bind to mRNA molecules of interest, followed by reversible or irreversible crosslinking of the mRNA molecules.

[0251] In some embodiments, the biological sample is immobilized in a hydrogel via cross-linking of the polymer material that forms the hydrogel. Cross-linking can be performed chemically and / or photochemically, or alternatively by any other suitable hydrogel-formation method. A hydrogel may include a macromolecular polymer gel including a network. Within the network, some polymer chains can optionally be cross-linked, although cross-linking does not always occur.

[0252] In some embodiments, a matrix includes matrix subunits. In some embodiments, the matrix is a hydrogel, and the hydrogel includes hydrogel subunits, such as, but not limited to, acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, poly(ethyleneny-2785575202412020440 glycol) and derivatives thereof (e.g. PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof. In some embodiments, a hydrogel can be formed using between about 1% and about 10% acrylamide and between about 0.01% and about 1% bis- acrylamide in a buffer such as SSC. In some embodiments, a hydrogel can be formed using between about 2% and about 6% acrylamide and between about 0.1% and about 0.5% bis- acrylamide in a buffer such as SSC. In some embodiments, a hydrogel can be formed using about 4% acrylamide and about 0.2% bis-acrylamide in a buffer such as 2X SSC. In some embodiments, the bis-acrylamide concentration can be increased to increase the degree of cross- linking between monomers to form a matrix such as a hydrogel. In some embodiments, the concentration of a crosslinker can be increased to reduce cracking of a hydrogel during sandwiching.

[0253] In some embodiments, a hydrogel includes a hybrid material, e.g., the hydrogel material includes elements of both synthetic and natural polymers. Examples of suitable hydrogels are described, for example, in U.S. Patent Nos. 6,391,937, 9,512,422, and 9,889,422, and in U.S. Patent Application Publication Nos. 2017 / 0253918, 2018 / 0052081 and 2010 / 0055733, the entire contents of each of which are incorporated herein by reference.

[0254] The composition and application of the hydrogel-matrix to a biological sample typically depends on the nature and preparation of the biological sample (e.g., sectioned, non-sectioned, type of fixation). As one example, where the biological sample is a tissue section, the hydrogel-matrix can include a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) accelerator solution. As another example, where the biological sample contains cells (e.g., cultured cells or cells disassociated from a tissue sample), the cells can be incubated with the monomer solution and APS / TEMED solutions. For cells, hydrogel-matrix gels are formed in compartments, including but not limited to devices used to culture, maintain, or transport the cells. For example, hydrogel-matrices can be formedny-2785575202412020440 with monomer solution plus APS / TEMED added to the compartment to a depth ranging from about 0.1 μm to about 2 mm.

[0255] Additional methods and aspects of hydrogel embedding of biological samples are described for example in Chen et al., Science 347(6221):543–548, 2015, the entire contents of which are incorporated herein by reference.

[0256] In some embodiments, the hydrogel can form the substrate. In some embodiments, the substrate includes a hydrogel and one or more second materials. In some embodiments, the hydrogel is placed on top of one or more second materials. For example, the hydrogel can be pre-formed and then placed on top of, underneath, or in any other configuration with one or more second materials. In some embodiments, hydrogel formation occurs after contacting one or more second materials during formation of the substrate. Hydrogel formation can also occur within a structure (e.g., wells, ridges, projections, and / or markings) located on a substrate.

[0257] In some embodiments, hydrogel formation on a substrate occurs before, contemporaneously with, or after probes are affixed to the substrate. For example, hydrogel formation can be performed on the substrate already containing the probes.

[0258] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., tissue section) is embedded in a hydrogel. In some embodiments, hydrogel subunits are infused into the biological sample, and polymerization of the hydrogel is initiated by an external or internal stimulus.

[0259] In embodiments in which a hydrogel is formed within a biological sample, functionalization chemistry can be used. In some embodiments, functionalization chemistry includes hydrogel-tissue chemistry (HTC). Any hydrogel-tissue backbone (e.g., synthetic or native) suitable for HTC can be used for anchoring biological macromolecules and modulating functionalization. Non-limiting examples of methods using HTC backbone variants include CLARITY, PACT, ExM, SWITCH and ePACT. In some embodiments, hydrogel formation within a biological sample is permanent. For example, biological macromolecules can permanently adhere to the hydrogel allowing multiple rounds of interrogation. In some embodiments, hydrogel formation within a biological sample is reversible. In some embodiments, HTC reagents are added to the hydrogel before, contemporaneously with, and / or after polymerization. In some embodiments, a cell labeling agent is added to the hydrogel before,ny-2785575202412020440 contemporaneously with, and / or after polymerization. In some embodiments, a cell-penetrating agent is added to the hydrogel before, contemporaneously with, and / or after polymerization.

[0260] In some embodiments, a method disclosed herein comprises contacting a biological sample (e.g., an FFPE tissue sample or a fresh frozen tissue sample) with an attachment agent configured to attach to a target nucleic acid in the biological sample. In some embodiments, the attachment agent is covalently or noncovalently attached to a 3’ end of the target nucleic acid (e.g., the 3’ terminal residue of an RNA). In some embodiments, the attachment agent is covalently or noncovalently attached to a 5’ end of the target nucleic acid (e.g., the 5’ terminal residue of an RNA). In some embodiments, the method further comprises embedding the biological sample in a matrix (e.g., a hydrogel matrix), wherein the attachment agent (which is attached to the target nucleic acid) is covalently or noncovalently attached to the matrix. In some embodiments, the biological sample is contacted with at least two different attachment agents: a first attachment agent configured to be covalently attached to the target nucleic acid, and a second attachment agent comprising an oligonucleotide that hybridizes to the target nucleic acid. In some embodiments, the second attachment agent comprises a poly(dT) sequence that hybridizes to a poly(A) sequence in a target RNA. In some embodiments, the second attachment agent comprises dT and / or locked dT. In some embodiments, the first and second attachment agents each comprises an acrydite moiety that covalently binds the matrix. In some embodiments, a matrix-forming agent (e.g., a hydrogel subunit) comprises an attachment agent configured to attach to a target nucleic acid in the biological sample, and the matrix- forming agent is contacted with a biological sample to allow attachment of the target nucleic acid, followed by forming the matrix (e.g., polymerization to form a hydrogel) to embed the biological sample and at the same time attach the attachment agent (which is attached to the target nucleic acid) to the matrix. In some embodiments, the method further comprises clearing at least some non-target cellular components from the matrix wherein the target nucleic acid remains tethered to the matrix. In some embodiments, the method further comprises releasing the target nucleic acid from the matrix. In some embodiments, the method further comprises hybridizing or ligating the released target nucleic acid to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode. In some embodiments, the method further comprises determining the sequence of all or a portion of theny-2785575202412020440 target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0261] In some embodiments, additional reagents are added to the hydrogel subunits before, contemporaneously with, and / or after polymerization. In some embodiments, a hydrogel subunit comprises an attachment agent configured to attach to a target nucleic acid in a biological sample and to attach to one or more other hydrogel subunits. For example, additional reagents can include but are not limited to oligonucleotides (e.g., probes), endonucleases to fragment DNA, fragmentation buffer for DNA, DNA polymerase enzymes, and the like. Other enzymes can be used, including without limitation, RNA polymerase, ligase, proteinase K, and DNAse. Additional reagents can also include reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers, and oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, contemporaneously with, and / or after polymerization.

[0262] Biological samples embedded in hydrogels can be cleared using any suitable method. For example, electrophoretic tissue clearing methods can be used to remove biological macromolecules from the hydrogel-embedded sample. In some embodiments, a hydrogel- embedded sample is stored before or after clearing of hydrogel, in a medium (e.g., a mounting medium, methylcellulose, or other semi-solid mediums). In some embodiments, the method comprises removing the biological sample from the matrix. In some embodiments, removing the biological sample comprises clearing the biological sample. In some embodiments, the method comprises removing a subset of cellular components from the biological sample in the matrix. In some embodiments, removing the subset of cellular components from the biological sample comprises clearing the biological sample.

[0263] In some embodiments, a biological sample embedded in a matrix (e.g., a hydrogel) can be isometrically expanded. Isometric expansion methods that can be used include hydration, a preparative step in expansion microscopy, as described in, e.g., Chen et al., Science 347(6221):543–548, 2015 and U.S. Pat. 10,059,990, all of which are herein incorporated by reference in their entireties. Isometric expansion of the sample can increase the spatial resolution of the subsequent analysis of the sample. The increased resolution in spatial profiling can be determined by comparison of an isometrically expanded sample with a sample that has not been isometrically expanded. In some embodiments, a biological sample is isometrically expanded tony-2785575202412020440 a size at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its non- expanded size. In some embodiments, the sample is isometrically expanded to at least 2x and less than 20x of its non-expanded size.

[0264] In some embodiments, a biological sample is embedded in a matrix and target nucleic acid, such as DNA and / or RNA, from the biological sample is tethered to the matrix. In some embodiments, embedding a biological sample comprises tethering a target RNA from the biological sample to a matrix. In some embodiments, a biological sample is embedded in a matrix prior to tethering target RNA from the biological sample to the matrix. In some embodiments, a target RNA from a biological sample is tethered to a matrix-forming agent prior to embedding of the biological sample in a matrix formed by the matrix-forming agent. In some embodiments, a target RNA from a biological sample is tethered to a matrix-forming agent contemporaneously with the polymerization of the matrx-forming agent to embed the biological sample in a matrix formed by the matrix-forming agent. In some embodiments, the matrix- forming agent comprises an attachment agent. In some embodiments, the embedding of the biological sample in the matrix comprises polymerization of subunits to form the matrix. In some embodiments, the matrix is a hydrogel matrix. In some embodiments, the matrix is a hydrogel matrix, and the matrix subunits are hydrogel subunits. (iii) Staining, Destaining, and Immunohistochemistry (IHC)

[0265] To facilitate visualization, biological samples can be stained using a wide variety of stains and staining techniques. In some embodiments, for example, a sample can be stained using any number of stains and / or immunohistochemical reagents. One or more staining steps may be performed to prepare or process a biological sample for an assay described herein or may be performed during and / or after an assay. In some embodiments, the sample can be contacted with one or more nucleic acid stains, membrane stains (e.g., cellular or nuclear membrane), cytological stains, or combinations thereof. In some examples, the stain may be specific to proteins, phospholipids, DNA (e.g., dsDNA, ssDNA), RNA, an organelle or compartment of the cell. The sample may be contacted with one or more labeled antibodies (e.g., a primary antibody specific for the analyte of interest and a labeled secondary antibody specific for the primary antibody). In some embodiments, cells in the sample can be segmented using one or more images taken of the stained sample.ny-2785575202412020440

[0266] In some embodiments, the stain is performed using a lipophilic dye. In some examples, the staining is performed with a lipophilic carbocyanine or aminostyryl dye, or analogs thereof (e.g, DiI, DiO, DiR, DiD). Other cell membrane stains may include FM and RH dyes or immunohistochemical reagents specific for cell membrane proteins. In some examples, the stain may include but is not limited to, acridine orange, acid fuchsin, Bismarck brown, carmine, coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, haematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, ruthenium red, propidium iodide, rhodamine (e.g., rhodamine B), or safranine, or derivatives thereof. In some embodiments, the sample may be stained with haematoxylin and eosin (H&E).

[0267] The sample can be stained using hematoxylin and / or eosin staining techniques, using Papanicolaou staining techniques, Masson’s trichrome staining techniques, silver staining techniques, Sudan staining techniques, and / or using Periodic Acid Schiff (PAS) staining techniques. PAS staining is typically performed after formalin or acetone fixation. In some embodiments, the sample can be stained using Romanowsky stain, including Wright’s stain, Jenner’s stain, Can-Grunwald stain, Leishman stain, and Giemsa stain.

[0268] In some embodiments, biological samples can be destained. Any suitable methods of destaining or discoloring a biological sample may be utilized and generally depend on the nature of the stain(s) applied to the sample. For example, in some embodiments, one or more immunofluorescent stains are applied to the sample via antibody coupling. Such stains can be removed using techniques such as cleavage of disulfide linkages via treatment with a reducing agent and detergent washing, chaotropic salt treatment, treatment with antigen retrieval solution, and treatment with an acidic glycine buffer. Methods for multiplexed staining and destaining are described, for example, in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6:8390, Pirici et al., J. Histochem. Cytochem. 2009; 57:567–75, and Glass et al., J. Histochem. Cytochem. 2009; 57:899–905, the entire contents of each of which are incorporated herein by reference. B. Nucleic Acid Tethering to Matrix

[0269] One advantage of tethering nucleic acids, such as DNA and / or RNA molecules, to the matrix is the ability to remove ribosomes and other proteins, such as those bound to the RNA molecules, to increase the available regions for probe hybridization. In some 74ny-2785575202412020440 embodiments, the tethering is reversible. In some embodiments, RNA molecules of a biological sample embedded in a matrix can be tethered to the matrix, followed by clearing the biological sample to remove ribosomes and / or other proteins. The RNA molecules can remain tethered during sample clearing, and the tethering can be reversed, thereby releasing the RNA molecules for subsequent analysis. In some embodiments, dissolvable gel formulations are used. For example, RNA molecules can be tethered to a hydrogel during sample clearing, wherein the hydrogel is dissolved to release the RNA molecules after sample clearing. Tethering of RNA molecules to a matrix can be achieved through any of the methods disclosed in US 2024 / 0218424, which is herein incorporated by reference in its entirety.

[0270] As detailed herein, the present disclosure relates in one aspect to the preparation of a ribonucleic acid (RNA) comprising a 5’-phosphate group or a 5’-phosphate group modified with a leaving group for immobilization of the ribonucleic acids in a matrix. In some embodiments, the methods provided herein achieve immobilization of the ribonucleic acids through the use of an attachment agent that mediates the interaction between the ribonucleic acid and the matrix-forming agent and ultimately the matrix. In some embodiments, the methods as provided herein comprise contacting the biological sample with one or more attachment agent. In some embodiments wherein the method comprises contacting the biological sample with two or more attachment agents, the attachment agents is the same or different.

[0271] In some embodiments, the attachment agent is a multifunctional molecule. In some embodiments, the attachment agent comprises at least one (e.g., 1, 2, 3, or 4) reactive moiety capable of covalently bonding to the ribonucleic acid and at least one (e.g., 1, 2, 3, or 4) attachment moiety capable of covalently or non-covalently bonding to a matrix-forming agent. It should be recognized that reference to the attachment agent, whether bi- or multifunctional, as used herein in some embodiments refers to the attachment agent prior to binding with the ribonucleic acid and the matrix-forming agent, unless otherwise noted.

[0272] In some embodiments, the attachment agent is a compound of Formula (I):(I), or a salt thereof, wherein each RRNAis independently a reactive moiety capable of reacting with at least one 5’-phosphate group of the RNA or 5’- phosphate group of the RNA modified with a leaving group; each RAMis independently anny-2785575202412020440 attachment moiety capable of attaching covalently or noncovalently to a matrix-forming agent; L is a bond or a linker moiety; m is an integer from 1 to 4; and p is an integer from 1 to 4.

[0273] In some embodiments, RRNA, RAM, L, m, and p are each as defined herein. It should be understood that every description, variation, embodiment or aspect of a moiety may be combined with every description, variation, embodiment or aspect of other moieties the same as if each and every combination of descriptions is specifically and individually listed. For example, every description, variation, embodiment or aspect provided herein with respect to RRNAof Formula (I) may be combined with every description, variation, embodiment or aspect of L of Formula (I) the same as if each and every combination were specifically and individually listed. For another example, every description, variation, embodiment or aspect provided herein with respect to RAMof Formula (I) may be combined with every description, variation, embodiment or aspect of L of Formula (I) the same as if each and every combination were specifically and individually listed. (i) Reactive moiety RRNA

[0274] In some embodiments, the attachment agent comprises a reactive moiety. As provided herein, the reactive moiety capable of covalently bonding to the ribonucleic acid can be any reactive moiety that reacts with and covalently bonds to a 5’-phosphate or a 5’-phosphate modified with a leaving group. In some variations, such reactive moiety capable of covalently bonding to the ribonucleic acid by covalently bonding to a 5’-phosphate on the ribonucleic acid. In some variations, such reactive moiety capable of covalently bonding to the ribonucleic acid by covalently bonding to a 5’-phosphate on the ribonucleic acid modified with a leaving group.

[0275] In some embodiments, at least one reactive moiety is capable of reacting with at least one 5’-phosphate group of the RNA via an enzymatic reaction. In some embodiments, at least one reactive moiety of the attachment agent comprises or is a nucleic acid oligonucleotide comprising between 2 to 30 (e.g., between any of 2 to 25, 2 to 20, 2 to 15, or 5 to 15) nucleotide residues. In some embodiments, the nucleic acid oligonucleotide is a DNA oligonucleotide. The nucleic acid oligonucleotide can comprise any nucleic acid sequence, (e.g., any sequence of nucleotide residues). In some embodiments, the nucleic acid oligonucleotide comprises a random sequence. In some embodiments, the nucleic acid oligonucleotide comprises at least one thymine (T). In some embodiments, the nucleic acid oligonucleotide comprises a sequence of at least 2, 3, 4, 5, 6, 7, 8, or more thymines. In some embodiments, the nucleic acid oligonucleotide does notny-2785575202412020440 comprise a sequence of thymines. In some embodiments, p is 4. In some embodiments, p is 3. In some embodiments, p is 2. In some embodiments, p is 1. In some embodiments, the attachment agent is of Formula (I-a):wherein DNA-1 comprises a nucleic acid sequence of 7-15 nucleotide residues; each RAMis independently an attachment moiety capable of attaching covalently or noncovalently to a matrix-forming agent; L is a bond or a linker moiety; and m is an integer from 1 to 4.

[0276] In some embodiments, the DNA-1 comprises at least one thymine (T). In some embodiments, tethering RNA to the matrix comprises forming a covalent bond between a 3’-OH of the DNA-1 and the 5’-phosphate group of the RNA under the catalysis of a ligase. In some embodiments, the ligase is an RNA ligase. In some embodiments, the ligase is T4 RNA Ligase 1.

[0277] In some embodiments, provided herein is a method comprising reacting a reactive moiety RRNAwith an RNA comprising a 5’-phosphate through an enzymatic process. In some embodiments, the attachment moiety RAMis an acrydite. In some embodiments, the reactive moiety RRNAis a nucleic acid, such as a DNA. In some embodiments, the DNA is a sequence of thymine residues. However, the nucleic acid oligonucleotide (e.g., DNA oligonucleotide) can comprise any sequence. In some embodiments, the nucleic acid oligonucleotide does not comprise a sequence of thymines. In some embodiments, the nucleic acid oligonucleotide comprises or is a random sequence. In some embodiments, the reactive moiety RRNA(e.g., a nucleic acid comprising a 3’ hydroxyl group) reacts with an RNA comprising a 5’-phosphate under the catalysis of an RNA ligase, such as T4 RNA Ligase 1. In the example illustrated, RRNAis a nucleic acid comprising 8 thymine (T).

[0278] In some embodiments, at least one reactive moiety is capable of reacting with at least one 5’-phosphate group or 5’-phosphate group modified with a leaving group of the RNA via a non-enzymatic reaction. In some embodiments, at least one reactive moiety is capable ofny-2785575202412020440 reacting with a 5’-phosphate group modified with a leaving group of the RNA via a non- enzymatic reaction, such a substitution reaction.

[0279] In some embodiments, at least one reactive moiety comprises or is a nucleophilic group capable of reacting with 5’-phosphate group modified with a leaving group of the ribonucleic acid. In some embodiments, at least one reactive moiety comprises or is an amine moiety, an amide moiety, an alcohol moiety, a thiol moiety, a cyano moiety, an ylide moiety, a hydrazide, a hydroxylamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, or an arylhydrazide, or any combination thereof. In some embodiments, at least one reactive moiety is or comprises an amine moiety, an amide moiety, an alcohol moiety, a thiol moiety, a cyano moiety, or an ylide moiety.

[0280] In some embodiments, at least one reactive moiety is or comprises an amine moiety (e.g., -NHR or -NH2), an alcohol moiety, or a thiol moiety that is capable of reacting with a 5’-phosphate group modified with a leaving group. In some embodiments, the reactive moiety of the attachment agent is or comprises an amine moiety (e.g., -NHR or -NH2). In some embodiments, the reaction of an amine moiety with a 5’-phosphate group modified with a leaving group of the ribonucleic acid forms a P-NH or -P-NR bond. In some embodiments, the reactive moiety of the attachment agent is or comprises an alcohol moiety (e.g., -OH). In some embodiments, the reaction of an alcohol moiety with a 5’-phosphate group modified with a leaving group of the ribonucleic acid forms a P-O bond. In some embodiments, the reactive moiety of the attachment agent is or comprises a thiol moiety (e.g., -SH). In some embodiments, the reaction of a thiol moiety with the 5’-phosphate group modified with a leaving group of the ribonucleic acid forms a P-S bond.

[0281] In some embodiments, the method comprises anchoring both a 5’ end and a 3’ end of an RNA to the biological sample or to a matrix embedding the biological sample. In some instances, the method comprises anchoring a 5’ end of an RNA to the biological sample or to a matrix embedding the biological sample according to any of the embodiments described herein, and further comprises anchoring the 3’ end of the RNA to the matrix. In some embodiments, anchoring the 3’ end of the RNA to the matrix comprises contacting the biological sample with a probe that hybridizes to the 3’ end of the RNA (e.g., a probe comprising a plurality of thymine bases that hybridizes to a polyA tail of the RNA). In some embodiments, anchoring the 3’ end of the RNA comprises contacting the biological sample comprising the RNA with a formylationny-2785575202412020440 reagent, wherein the RNA comprises a 2’,3’-vicinal diol and the formylation reagent converts the 2’,3’-vicinal diol moiety into a 2’,3’-dialdehyde moiety; and contacting the biological sample with a 3’-end attachment agent comprising at least one aldehyde-reactive group capable of reacting with at least one aldehyde of the 2’,3’-dialdehyde moiety of the ribonucleic acid to form a covalent bond and an attachment moiety capable of attaching covalently or non-covalently to an exogenous or endogenous molecule in the biological sample (e.g., to a matrix).

[0282] In some embodiments, the 3’-end attachment agent comprises an aldehyde- reactive group. In some embodiments, the aldehyde-reactive group comprises or is a nucleophilic group capable of reacting with at least one aldehyde of the 2’,3’- dialdehyde moiety of the ribonucleic acid. In some embodiments, the reactive group comprises or is an amine moiety, an amide moiety, an alcohol moiety, a thiol moiety, a cyano moiety, an ylide moiety, a hydrazide, a hydroxylamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, or an arylhydrazide, or any combination thereof. In some embodiments, the aldehyde-reactive group or first reactive group of the attachment agent is or comprises an amine moiety, an amide moiety, an alcohol moiety, a thiol moiety, a cyano moiety, or an ylide moiety. In some embodiments, the aldehyde- reactive group of the 3’-end attachment agent is or comprises an amine moiety (e.g., -NHR or - NR2). In some embodiments, the reaction of an amine moiety with an aldehyde moiety of the ribonucleic acid forms an imine or an enamine. In some embodiments, the method comprises reduction of the imine (e.g., with NaBH4, optionally with 0.2M NaBH4).

[0283] In some instances, the biological sample is contacted with a TN4 PNK to polish the 3' ends into diols and add 5' phosphates to the RNAs. In some embodiments, after T4 PNK polishing, the method comprises incubating the biological sample with a ligase and an attachment agent according to any of the embodiments of Formula (I-a) described herein (e.g., a tethering oligonucleotide comprising a 5’ acrydite). In some embodiments, the biological sample is contacted with between about 10 nM and about 100 nM of the tethering oligonucleotide. In some embodiments, the biological sample is contacted with about 20 nM of tethering oligonucleotide. In some embodiments, the method comprises incubating the biological sample with the tethering oligonucleotide, ligase, and a PEG reagent (e.g., PEG8000) at a temperature of about 25°C for about 1 hour to about 4 hours (e.g., at least about 1, 1.5, or 2 hours). In some embodiments, the method further comprises tethering the polished 3’ end of the RNA by RNA oxidation (e.g., with NaIO4, optionally with 20mM NaIO4), aldehyde coupling (e.g., with 2AEM,ny-2785575202412020440 methacrylamide, Et3N, and aniline), and imine reduction (e.g., with NaBH4, optionally with 0.2M NaBH4).

[0284] In some embodiments, the 5’-phosphate group modified with a leaving group are contacted with an attachment agent comprising an amine moiety (e.g., -NH2), an alcohol moiety, or a thiol moiety, to form a link between the attachment agent and the RNA.

[0285] In some embodiments, the resulting 5’-phosphate group is further modified with a leaving group, and thereby forming an RNA comprising a 5’-phosphate group modified with a leaving group. In some embodiments, the modification comprise replacing one -OH group in the 5’-phosphate group with a leaving group. In some embodiments, the conjugation acid of the leaving group has a pKa of less than 8, such as less than about any of 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, or 3. In some embodiments, the leaving group is any one of Cl, Br, I, -OR, -OC(O)R, -OS(O)2R, or -NR1R2, wherein each R is independently haloalkyl, phenyl substituted with one or more alkyl or haloalkyl, or heteroaryl substituted with one or more alkyl or haloalkyl, and wherein R1is independently H, alkyl, or haloalkyl, R2is independently haloalkyl, phenyl substituted with one or more alkyl or haloalkyl, or heteroaryl substituted with one or more alkyl or haloalkyl, or R1and R2are taken together the N atom to which they are attached to form a heteroaryl. In some embodiments, the leaving groupwherein the wavy line denotes the attachment of the leaving group to the 5’ phosphate of the RNA.

[0286] In some embodiments, the step of contacting the biological sample comprising an RNA and attachment agent further comprises contacting the biological sample with one or more reagents or under suitable conditions to facilitate the formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and the reactive moiety of the attachment agent. For example, in some embodiments, the methods provided herein comprise contacting the attachment agent and the biological sample with aniline and / or triethylamine to facilitate formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and the reactive moiety of the attachment agent. In some embodiments, the methods comprise contacting the attachment agent and the biological sample with triethylamine. In some embodiments, the methods comprise contacting theny-2785575202412020440 attachment agent and the biological sample with triethylamine. In some embodiments, the methods do not comprise contacting the attachment agent and the biological sample with triethylamine to facilitate formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and the reactive moiety of the attachment agent. In some embodiments, the formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and optionally the reactive moiety of the attachment agent is further facilitated by an inorganic salt. In some embodiments, the inorganic salt is MgCl2. In some embodiments, the formation of a covalent bond between the 5’-phosphate group modified with a leaving group of the ribonucleic acid and the reactive moiety of the attachment agent is facilitated by shifting the equilibrium to the right, optionally by removing the products after the substitution reaction. For example, in some embodiments when the leaving group isthe reaction is facilitated by selectively removing 1H-imidazole from the reaction system. (ii) Boronic Acid Moiety

[0287] In some embodiments, RNA tethering to the matrix uses an attachment agent comprising boronic acid moieties in the matrix to passively capture 3’ RNA ends that have been polished, where the tethering occurs when the pH is greater than the pKa of the boronic acid moiety (which induces the formation of boronate esters with the 3’ RNA diols) and the tethering is reversed when the pH is below than the pKa.

[0288] In some embodiments, a matrix (e.g., a hydrogel) and / or a matrix-forming agent comprising a boronic acid moiety can be used to tether an RNA of a sample (e.g., a cell or tissue sample embedded in the hydrogel) for subsequent removal of proteins (e.g., ribosome components) and lipids. In some embodiments, the hydrogel is sandwiched into a non-boronic acid hydrogel holding an array. The array embedded in the hydrogel can be generated by inverting an array in a 5’ up configuration into a matrix (e.g., a non-boronic acid hydrogel which can be non-reversible), such that the inverted array is in a 3’ up configuration and is embedded in the hydrogel. The inversion can help remove incorrectly synthesized molecules and introduce a more enzymatically friendly environment for downstream analysis, for instance, when a sample may need to interface with the oligonucleotides (e.g., capture probes) on the array. The oligonucleotides (e.g., capture probes) can be provided in a pattern on the array substrate, such as 81ny-2785575202412020440 in array spots. A drop in pH below the pKa of the boronic acid moieties in the tethered sample can release the RNAs on command to begin interfacing with the array oligonucleotides on the underlying hydrogel. In some embodiments, the tethered RNA is pressed against the array in the presence of a slightly acidic buffer to un-tether the RNA, where the proximity of the array oligonucleotide helps limit lateral diffusion through the hydrogels to mitigate mis-localization. In some embodiments, capacitance can be used to drive migration of the newly liberated RNA towards the array. For instance, thin copper plates connected to a power source can leverage capacitance to drive migration of the newly untethered RNA towards the oligonucleotides on the array. As capacitance is a function of the area between the two plates and the distance between them, the RNA migration can be tuned to add a degree of control over the un-tethered RNA. In some embodiments, a conductive coating (e.g., Indium Tin Oxide) on both sides of the sandwich can be used to permit electrophoresis of the RNA onto the array. For instance, the inverted array (in a 3’ up configuration) and the boronic acid hydrogel can be deposited on a methacrylic acid coated oxide surface, including but not limited to silicon oxide or quartz. A conductive coating that conducts current can be used to actively electrophorese the untethered RNA onto the array, providing control of RNA migration and / or capture using voltage, amperage, ionic conditions in the buffer, and / or temperature.

[0289] In some embodiments, a boronic acid-based hydrogel can be used as a sample hydrogel for embedding a sample and a standard hydrogel can be used as an array hydrogel. In some embodiments, boronic acids may be embedded within the sample hydrogel matrix (e.g., a hydrogel mesh), to passively capture 3’ RNA ends that have been polished. In some approaches, the tethering of the RNA target molecules occurs if the pH of the system is greater than the pKa of the boronic acid. In some embodiments, this may be due to the formation of boronate esters between the boronic acid groups of the sample hydrogel with the 3’ RNA diols of the target RNA molecule. For embodiments, where the pH is below the pKa tethering may be revered and the tethered RNA target molecule can be released. In such an embodiment, no separate linker molecule is required.

[0290] In some embodiments, two different hydrogels may be prepared: a boronic acid hydrogel (e.g., sample hydrogel) and a standard hydrogel (e.g., non-reversible hydrogel, non-boronic acid hydrogel, array hydrogel, or reader hydrogel). In some embodiments, capture probes (e.g., capture agents) of a spatial array are intermeshed in the standard hydrogel. Theny-2785575202412020440 sandwich arrangement, in accordance with some embodiments may include the boronic acid hydrogel-based reversible tethering scheme as depicted in FIG. 20A.

[0291] In some embodiments, the boronic acid hydrogel may be formed over the tissue, where the RNA is tethered to the hydrogel. In some embodiments, a clearing step may be used to remove the ribosomes and / or prepare the sample to interface with the (non-boronic acid) hydrogel containing a spatial array for capturing nucleic acid molecules from the sample hydrogel.

[0292] In some embodiments, the sample hydrogel may be sandwiched into the standard hydrogel. In some embodiments, the tethered RNA may be pressed against the capture array in the presence of a slightly acidic buffer, allowing the target RNA to be released from the sample hydrogel. In some embodiments, due to the proximity of the capture probes (e.g., array oligos), lateral diffusion may be negligible as the target RNA diffuses through both the sample hydrogel and the capture hydrogel, as depicted in FIG. 20A, thereby mitigating mis-localization.

[0293] In some examples, pH triggered release of target molecules (e.g., target nucleic acid, target RNA, target RNA molecules) tethered to a hydrogel may be followed by diffusion of the mRNA target to the capture array. In some examples, capacitance may be utilized to limit diffusion in one direction (e.g., anisotropic diffusion), where released target molecules are directed toward the capture array, thereby increasing the efficiency of the capture process. In some embodiments, an external field driving the electrophoretic migration of the target molecule to the capture array (e.g., hydrogel barcoded capture array) is provided by thin copper plates connected to a power source as depicted in FIG. 20B where the copper plates act as or are connected to a cathode and an anode, respectively. The capacitance, C, of a pair of plates of a given material can be described as a function of three parameters: the plate area, A; the distance, d, between the two plates and the dielectric constant, ed, of the material from which the plates are made as shown in Function 1: C = ede0A / 2d.

[0294] In some embodiments, use of thin conductive layers applied to both the sample substrate and the capture array substrate may provide a capacitance allowing for electrophoretic migration as described in the current application. FIG. 20C depicts a thin film of indium tin oxide (ITO) deposited on both the sample substrate and the capture array substrate in accordance with some embodiments. In some embodiments, the spatial array hydrogel and theny-2785575202412020440 boronic acid hydrogel are deposited on a methacrylic acid coated, conductive oxide surface. In some embodiments, the oxide surface may comprise ITO. In some embodiments, the conductive oxide surface may be used provide a capacitive force allow for electrophoretic migration of released target molecules the hydrogel barcoded capture array (e.g., ITO coated substrate with capture probes). ITO coated substrates may allow for a great degree of control in the release and electrophoretic driven migration (e.g., directionally directed, or anisotropic diffusion), as voltage, amperage, ionic conditions in the buffer and temperature may be optimized and controlled. (iii) Attachment Moiety

[0295] In some embodiments, the attachment agent comprises an attachment moiety. In some embodiments, an attachment moiety is any functional group that interacts with a matrix- forming agent and, in some embodiments, the attachment moiety comprises or is a group capable of reacting with, covalently binding, or non-covalently binding to a complementary reactive group on the matrix-forming agent.

[0296] In some embodiments, at least one attachment moiety is capable of attaching covalently to a matrix-forming agent. In some embodiments, at least one attachment moiety is or comprises an alkenyl, alkynyl, allyl or vinyl moiety, ally ester moiety, an acrylamide moiety, an amide moiety, an alcohol moiety, a polyol moiety, a furan moiety, a maleimide moiety, a norbornene moiety, a thiol moiety, a sulfide moiety, a phenol moiety, a urethane moiety, a cyano moiety, an amino moiety, an isocyanate moiety, an isothiocyanate moiety, an ether moiety, a dextran moiety, or an alginate moiety.

[0297] In some embodiments, at least one attachment moiety comprises or is an electrophilic group that is capable of interacting with a reactive nucleophilic group present on the matrix-forming agent to provide a covalent bond between the attachment moiety and the matrix- forming agent. In some embodiments, the nucleophilic groups on the matrix-forming agent having that capability include but are not limited to, sulfhydryl, hydroxyl and amino functional groups. In some embodiments, at least one attachment moiety comprises or is a maleimide, haloacetamide, or NHS ester.

[0298] In some embodiments, at least one attachment moiety comprises or is a nucleophilic group that is capable of interacting with a reactive electrophilic group present on the matrix-forming agent to provide a covalent bond between the attachment moiety and the matrix- forming agent. In some embodiments, at least one attachment moiety comprises or is a thiol,ny-2785575202412020440 phenol, amino, hydrazide, hydroxylamine, hydrazine, thiosemicarbazone, hydrazine carboxylate, or arylhydrazide. In some embodiments, each attachment moiety is independently or independently comprises a phenol moiety, an alkyne moiety, a norbornene moiety, a sulfide moiety, a furan moiety, a maleimide moiety, or an allyl ester moiety.

[0299] In some embodiments, at least one attachment moiety comprises or is a click functional group. Suitable click functional groups include functional groups compatible with a nucleophilic addition reaction, a cyclopropane-tetrazine reaction, a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, an alkyne hydrothiolation reaction, an alkene hydrothiolation reaction, a strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, an inverse electron- demand Diels-Alder (IED-DA) reaction, a cyanobenzothiazole condensation reaction, an aldehyde / ketone condensation reaction, and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. In some embodiments, the attachment moiety(ies) or second reactive group comprise or is any functional group involved in click reactions. In some embodiments, such click reactions involve (i) azido and cyclooctynyl; (ii) azido and alkynyl; (iii) tetrazine and dienophile; (iv) thiol and alkynyl; (v) cyano and amino thiol; (vi) nitrone and cyclooctynyl; or (vii) cyclooctynyl and nitrone. It should be recognized that in instances in which the attachment moiety comprises or is a click functional group, the matrix-forming agent to which it is capable of forming a covalent bond comprises the complementary click functional group to that of the attachment moiety. For example, in some embodiments, the attachment moiety comprises or is an azide moiety and the matrix-forming agent comprises a complementary alkyne moiety, or vice versa.

[0300] In some embodiments, at least one attachment moiety comprises or is a group capable of reacting with a matrix-forming agent. As detailed herein, examples of matrix-forming agents include but are not limited to acrylamide, bis-acrylamide, polyacrylamide and derivatives thereof, poly(ethylene glycol) and derivatives thereof (e.g. PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohols, polypropylene glycol, polytetramethylene oxide, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and the like, and combinations thereof. In some embodiments, at least oneny-2785575202412020440 attachment moiety comprises or is an alkenyl, allyl or vinyl moiety, an amide moiety, an alcohol moiety, a polyol moiety, a furan moiety, a maleimide moiety, a norbornene moiety, a thiol moiety, a phenol moiety, a urethane moiety, a cyano moiety, an isocyanate moiety, an isothiocyanate moiety, an ether moiety, a dextran moiety, or an alginate moiety. In some embodiments, at least one attachment moiety comprises or is an alkenyl, allyl or vinyl moiety (e.g., -C=C- or HC=C- or HC=C-CH2-), such as in N-(2-aminoethyl)methacrylamide, 2- aminoethyl methacrylate, 2-aminoethyl (E)-but-2-enoate, 2-aminoethyl methacrylate or methylacrylamide, or norbornene. Such alkenyl, allyl or vinyl moieties may be suitable for reaction with matrix-forming agents.

[0301] In some embodiments, at least one attachment moiety comprises or is an acrylate moiety, methacrylate moiety, acrylamide moiety, methacrylamide moiety, biotinyl moiety, dextrin moiety, a click moiety, a thiol moiety, norbornenyl moiety, furanyl moiety, alkyl ester moiety, or maleimidyl moiety. In certain embodiments, at least one attachment moiety comprises or is a biotinyl moiety, dextrin moiety, a click moiety, a thiol moiety, norbornenyl moiety, furanyl moiety, alkyl ester moiety, or maleimidyl moiety.

[0302] In some embodiments, the attachment agent is of Formula (I-b):wherein RRNAis any of the reactive moiety provided herein; wherein L is a bond or any of the linker moiety provided herein; wherein W is independently H or C1-6alkyl; wherein Y is H or C1-6 alkyl; and wherein X is NH, N(C1-6alkyl), or O.

[0303] In some embodiments, the attachment agent is of Formula (I-c):wherein n is an integer from 0 to 50; wherein Z is CH2or O;ny-2785575202412020440 m is an integer from 1 to 4; p is an integer from 1 to 4; wherein RRNAis any of the reactive moiety provided herein; and wherein RAMis phenol, azide, alkyne, norbornene, sulfide, furan, maleimide, or allyl ester.

[0304] In some embodiments, the formation of a covalent or non-covalent bond between the attachment moiety and the matrix-forming agent is mediated by an external reagent or stimulus. For example, in some embodiments, the formation of a covalent or non-covalent bond between the attachment moiety and the matrix-forming agent is initiated or induced by an enzyme, a catalyst, chemical reagents (e.g., acid, base, reducing agent, oxidant, etc.), heat, and / or light. In some embodiments, a covalent bond is formed between the attachment moiety and the matrix-forming agent. In some embodiments, the step of contacting the biological sample and attachment agent further comprises contacting the biological sample with one or more reagents or under suitable conditions to facilitate the formation of a covalent bond between at least one attachment moiety of the attachment agent and the matrix-forming agent. For example, in some embodiments wherein the attachment moiety comprises an alkene or a click functional group, the method optionally further comprises adding reagents to activate the alkene or click functional group, such as a radical initiator or a copper catalyst, respectively. In other embodiments wherein at least one attachment moiety comprises an alkenyl, allyl or vinyl moiety, the method optionally further comprises exposing the biological sample and attachment agent to (ultraviolet) light or heat to facilitate formation of a covalent bond. In some embodiments wherein at least one attachment moiety comprises or is a norbornene moiety, furan moiety, maleimide moiety, or other alkenyl, allyl or vinyl moiety, the method optionally further comprises exposing the sample to light or heat. In yet other embodiments, the method may further comprise adding an enzyme to facilitate formation of a covalent bond. For example, in some embodiments wherein at least one attachment moiety comprises or is a phenol moiety, the method optionally further comprises adding horseradish peroxidase (HRP).

[0305] In some embodiments, the attachment moiety is capable of attaching non- covalently to a matrix-forming agent. In some embodiments, the attachment moiety is capable of attaching non-covalently to an exogenous molecule in the biological sample. In some embodiments, the attachment moiety is capable of attaching non-covalently to an endogenous molecule in the biological sample. In some embodiments, the attachment moiety comprises or isny-2785575202412020440 a group capable of binding to a matrix-forming agent via non-covalent interaction, such as but not limited to hydrogen bonding, van der Waals interaction, and / or pi-stacking.

[0306] In some embodiments, the attachment agent is biotinylated. In some embodiments, the attachment moiety is a biotin moiety or a derivative thereof. (iv) Linker L

[0307] In some embodiments, the attachment agent comprises a linker and / or linker moiety. In some embodiments, such as some embodiments of Formula (I), (I-a), or (I-b), L is a bond. In some embodiments of Formula (I), L is a linker moiety. In some embodiment, L comprises or is an unbranched or branched C1-C150alkylene, which can be interrupted by 1 to 50 independently selected O, NH, N, S, C6-C12 arylene, or 5- to 12-membered heteroarylene. In some embodiments, L comprises or is an unbranched and uninterrupted C1-C150 alkylene. In some embodiments, L comprises or is a branched and uninterrupted C1-C150alkylene. In some embodiments, L comprises or is an unbranched C1-C150 alkylene interrupted by 1 to 50 NH, O, or S. In some embodiments, L comprises or is, Z is CH2, O, S; or NH; and n is an integer between 0 and 50. In some embodiments, L is, Z is CH2, O, S; or NH; and n is an integer between 0 and 50 (e.g., between 0 and 30, between 0 and 20, between 0 and 10, or any of 4, 5, 6, 7, 8, 9). In some embodiments, L comprises or is, Z is CH2, O, S; or NH; and n is an integer between 1 and 10. In some embodiment, L comprises or is, Z is CH2, O, S; or NH; and n is 6. In some embodiments, L comprises or is an unbranched C1-C150alkylene interrupted by 1 to 50 oxygen. In some embodiments, L comprises a polyethylene glycol portion or is a polyethylene glycol moiety. In some embodiments, L comprises or is, and n is an integer between 0 and 50. In some embodiments, L comprises or is,ny-2785575202412020440 and n is an integer between 1 and 10. In some embodiment, L is, and n is 6. In some embodiment, L is, and n is an integer between 0 and 50 (e.g., between 0 and 30, between 0 and 20, between 0 and 10, or any of 4, 5, 6, 7, 8, 9). In some embodiments, L comprises an oligoethylene glycol. In some embodiments, L is an oligoethylene glycol moiety. In some embodiments, L comprises or is a branched C1-C150alkylene interrupted by 1 to 50 oxygen. In some embodiments, L comprises or is an unbranched C1-C150 alkylene interrupted by 1 to 50 sulfurs. In some embodiments, L comprises or is, and 30, between 0 and 20, between 0 and 10, or any of 4, 5, 6, 7, 8, 9). In some embodiments, L comprises or is a branched C1-C150 alkylene interrupted by 1 to 50 sulfurs. In some embodiments, L comprises or is a branched C1-C150 alkylene interrupted by 1 to 50 -NH-. In some embodiments, L comprises or is an unbranched C1-C150alkylene interrupted by 1 to 50 - NH-. In some embodiments, L comprises or is, and n is an integer between 0 and 50. In some embodiment, L is, and n is 6. In some embodiment, L is, and n is an integer between 0 and 50 (e.g., between 0 and 30, between 0 and 20, between 0 and 10, or any of 4, 5, 6, 7, 8, 9). In some embodiments, L is a branched C1-C150 alkylene interrupted by 1 to 50 -NH-, wherein the -NH- is not at a branching point. In some embodiments, L comprises or is a branched C1-C150 alkylene interrupted by 1 to 50 -N-, wherein the -N- is at a branching point. In some embodiments, Lny-2785575202412020440 comprises or is an unbranched or branched C1-C150 alkylene interrupted by 1 to 50 independently selected C6-C12arylene, for example, any of phenyl or naphthalene. In some embodiments, L comprises or is an unbranched or branched C1-C150 alkylene interrupted by 1 to 50 independently selected 5- to 12-membered heteroarylene, for example, any of pyridine, furan, pyrrole, or thiophene. (v) Exemplary Formulae

[0308] In some embodiments, the attachment agent as described herein (e.g., the attachment agent of Formula (I)) comprises any of 1, 2, 3, or 4 reactive groups (e.g., RRNA). The reactive groups RRNA of Formula (I) are each independently selected and as defined in Section (B) (i) above. In some embodiments, the attachment agent is a bifunctional molecule comprising one reactive group. In some embodiments of Formula (I), p is any of 1, 2, 3, or 4. In some embodiments, the attachment agent (e.g., Formula (I)) comprises more than one reactive groups RRNA(e.g., p is any of 2, 3, or 4), wherein the RRNAgroups can be the same group, selected from the embodiments provided herein. In some embodiments, the attachment agent (e.g., Formula (I)) comprises more than one reactive groups RRNA(e.g., p is any of 2, 3, or 4), wherein each RRNAis independently selected from the embodiments provided herein, provided the more than one reactive groups are chemically compatible and have chemically compatible ribonucleic- binding mechanisms or reactions.

[0309] In some embodiments, the attachment agent (e.g., Formula (I)) comprises any of 1, 2, 3, or 4 attachment moieties (e.g., RAM). The attachment moieties RAMof Formula (I) are each independently selected and as defined in Section (B)(iii) above.

[0310] In some embodiments, RAMis capable of reacting with a matrix-forming agent to form a covalent bond. In some embodiments, RAMis capable of reacting with a matrix- forming agent. In some embodiments, RAMis In some embodiments, RAMis an alkenyl, allyl or vinyl moiety, an amide moiety, an alcohol moiety, a polyol moiety, a furan moiety, a maleimide moiety, a norbornene moiety, a thiol moiety, a phenol moiety, a urethane moiety, a cyano moiety, an isocyanate moiety, an isothiocyanate moiety, an ether moiety, a dextran moiety, or an alginate moiety. In some embodiments, RAMis a biotin moiety or a derivative thereof. In some embodiments, RAMis an acrylate moiety, methacrylate moiety, acrylamide moiety, methacrylamide moiety, biotinyl moiety, dextrin moiety, a click moiety, a thiol moiety, norbornenyl moiety, furanyl moiety, alkyl ester moiety, or maleimidyl moiety. In certainny-2785575202412020440 embodiments, RAMis a biotinyl moiety, dextrin moiety, a click moiety, a thiol moiety, norbornenyl moiety, furanyl moiety, alkyl ester moiety, or maleimidyl moiety.

[0311] In some embodiments of Formula (I), m is any of 1, 2, 3, or 4. In some embodiments, the attachment agent (e.g., Formula (I)) comprises more than one attachment moieties RAM(e.g., m is any of 2, 3, or 4), wherein the attachment moieties are the same group, selected from the embodiments provided herein. In some embodiments, the attachment agent (e.g., Formula (I)) comprises more than one attachment moieties RAM(e.g., m is any of 2, 3, or 4), wherein each RAMis independently selected from the embodiments provided herein, provided the more than one reactive groups are chemically compatible and their binding mechanism or reactions to the matrix-forming agent are also chemically compatible.

[0312] In some embodiments, the attachment agent is a compound of Formula (I-d),or a salt thereof, wherein W is independently H or C1-6 alkyl (e.g., CH3); Y is H or C1-6 alkyl (e.g., CH3); X is NH, N(C1-6alkyl), or O; Z is CH2, O, or S; n is an integer from 0 to 50 (e.g., an integer between 1 and 20 or between 1 and 10); and DNA-1 comprises a nucleic acid sequence of 7-15 nucleotide residues.

[0313] In some embodiments, the attachment agent is a compound of Formula (I-e),or a salt thereof, wherein W is independently H or C1-6alkyl (e.g., CH3); Y is H or C1-6 alkyl (e.g., CH3);ny-2785575202412020440 X is NH, N(C1-6 alkyl), or O; Z is CH2, O, or S; n is an integer from 0 to 50 (e.g., an integer between 1 and 20 or between 1 and 10); and

[0314] In some embodiments, the attachment agent is of Formula (I-f):wherein n is an integer from 0 to 50 (e.g., an integer between 1 and 20 or between 1 and 10); wherein Z is CH2or O; wherein RRNAis NH2 or OH; and wherein RAMis phenol, azide, alkyne, norbornene, sulfide, furan, maleimide, or allyl ester.

[0315] In some embodiments of any of Formulae (I-c), (I-d), (I-e), and (I-f), L is , Z is O and n is 6. In some embodiments wherein L is, L is a hexaethylene glycol moiety. C. Spatial Profiling of RNA

[0316] In some embodiments, provided herein is a method for spatial analysis of biological molecules in a cell or tissue sample on a substrate (e.g., a slide or slip such as a glass or plastic slide or slip), comprising tethering the biological molecules in the cell or tissue sample to a matrix (e.g., a hydrogel), followed by releasing the tethered biological molecules for subsequent analysis. In some embodiments, the biological molecules comprise nucleic acid molecules (e.g., RNA such as mRNA). In some embodiments, the cell or tissue sample is a fresh frozen sample or a formalin-fixed paraffin-embedded sample, such as a fresh frozen tissue section or a formalin-fixed paraffin-embedded tissue section on a substrate. In some embodiments, the method comprises embedding the fresh frozen sample or the formalin-fixed paraffin-embedded sample on a sample substrate in a hydrogel, and tethering nucleic acid 92ny-2785575202412020440 molecules (e.g., RNA such as mRNA) in the sample to the hydrogel during or after the formation of the hydrogel embedding the sample on the sample substrate. In some embodiments, a formalin-fixed paraffin-embedded sample is decrosslinked prior to embedding the sample in the hydrogel, and the decrosslinking may allow more RNA molecules to interact with the hydrogel matrix for tethering (e.g., RNA molecules previously crosslinked with cellular components such as proteins or lipids may be decrosslinked such that they can be tethered to the hydrogel matrix). In some embodiments, a formalin-fixed paraffin-embedded sample is decrosslinked after embedding the sample in the hydrogel, and decrosslinking the hydrogel-embedded sample may decrosslink RNA from cellular components such as proteins or lipids while allowing the RNA to remain tethered (via the 3’ end or 5’ end) to the hydrogel.

[0317] In some embodiments, at least one or more nucleic acid molecules (e.g., RNA such as mRNA) in the sample are 3’ tethered to the hydrogel. In some embodiments, at least one or more nucleic acid molecules (e.g., RNA such as mRNA) in the sample are 5’ tethered to the hydrogel. In some embodiments, the 3’ tethering and / or the 5’ tethering comprises use of an attachment agent. In some embodiments, the 3’ tethering and / or the 5’ tethering are reversed to allow release of the nucleic acid molecules (e.g., RNA such as mRNA) from the matrix on the sample substrate. In some embodiments, the released nucleic acid molecules (e.g., RNA such as mRNA) are captured by capture probes on an arrayed substrate of capture probes. In some embodiments, the sample substrate and the array substrate are the same substate. In some embodiments, the sample substrate and the array substrate are separate substates which can be brought into physical proximity to each other to facilitate the release, migration, and / or capture of the nucleic acid molecules. In some embodiments, a method disclosed herein is used for spatial transcriptomic profiling of an FF tissue section or an FFPE tissue section mediated by 3’ or 5’ target (e.g., nucleic acid molecules such as RNA, e.g., mRNA, or ligation products that serve as proxies of target RNA) tethering to a hydrogel.

[0318] In some embodiments, provided herein is a method for spatial analysis of a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target nucleic acid is tethered to a matrix of the hydrogel; b) digesting and removing the embedded biological sample from the hydrogel; c) releasing the target nucleic acid from the matrix of the hydrogel; d) hybridizing the released target nucleic acid to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcodeny-2785575202412020440 associated with the location of the capture probe on the array; and e) determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, thereby analyzing the location of the target nucleic acid in the biological sample. In some embodiments, the target nucleic acid is a cellular nucleic acid, such as genomic DNA or mRNA. In some embodiments, the target nucleic acid is a product of a cellular nucleic acid (e.g., genomic DNA or mRNA), the product can be generated in situ at a location in the biological sample (e.g., prior to digesting and removing the embedded biological sample from the hydrogel), and the product can be generated before, during, or after embedding the biological sample in the hydrogel. In some embodiments, the product can be a cDNA of an RNA molecule in the biological sample. In some embodiments, the target nucleic acid is a nucleic acid probe that hybridizes to a cellular nucleic acid (e.g., genomic DNA or mRNA) or a nucleic acid probe that hybridizes to a product (e.g., cDNA) of the cellular nucleic acid. In some embodiments, the target nucleic acid is a product of nucleic acid probes that hybridize to a cellular nucleic acid (e.g., genomic DNA or mRNA) or a product of nucleic acid probes that hybridize to a product (e.g., cDNA) of the cellular nucleic acid. In some embodiments, the product of the nucleic acid probes is a ligation product, which can be analyzed as proxy of the cellular nucleic acid. In some embodiments, the array comprises a substrate with capture probes immobilized thereon in array features and a hydrogel on the substrate. In some embodiments, the array comprises a substrate with capture probes immobilized thereon in array features and no hydrogel on the substrate.

[0319] In some embodiments, provided herein is a method for analyzing a biological sample comprising tethering a target RNA to a hydrogel matrix, clearing the biological sample, and releasing the target RNA from the hydrogel matrix. In some embodiments, provided herein is a method for determining a spatial transcriptomic profile for a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target RNA from the biological sample is tethered to a matrix of the hydrogel; b) digesting and removing the embedded biological sample from the hydrogel; c) applying a first and a second probe to the hydrogel, wherein the first probe comprises a functional sequence and a sequence substantially complementary to a first portion of the target RNA and the second probe comprises a sequence substantially complementary to a second portion of the target RNA and a capture domain that is substantially complementary to a capture domain of a capture probe on an array, wherein theny-2785575202412020440 capture probe further comprises a spatial barcode; d) hybridizing the first probe and the second probe to the target RNA tethered to the matrix of the hydrogel; e) ligating together the first probe and the second probe hybridized to the target RNA in the hydrogel, thereby generating a ligation product that is a proxy of the target RNA; f) releasing the ligation product from the target RNA in the hydrogel and hybridizing the released ligation product to the capture domain of the capture probe on the array; g) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the ligation product, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the array comprises a substrate with capture probes immobilized thereon in array features and a hydrogel on the substrate. In some embodiments, the array comprises a substrate with capture probes immobilized thereon in array features and no hydrogel on the substrate.

[0320] Turning to FIG. 12A, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. an FFPE sample) with the steps of 1) baking the slide comprising the sample; 2) dewaxing the sample; 3) rehydrating the sample; 4) performing H&E staining; 5) imaging the sample (e.g. using brightfield (BF) imaging); and / or 6) destaining the sample (1201). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1202). In some embodiments, the method further comprises tethering a target (e.g., a target nucleic acid such as RNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1203). In some embodiments, the method further comprises decrosslinking the sample in the matrix (1204). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while tethered target remains attached to matrix (1205). In some embodiments, the method further comprises contacting the tissue sample with a probe or probe set that directly or indirectly binds to the target tethered to the matrix, followed by one or more optional wash(es) (1206). In some embodiments, the method further comprises ligating the probe or probe set bound to the target within the matrix (e.g., RNA-templated ligation), followed by one or more optional wash(es) (1207). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared tissue sample (e.g., a “tissue” hydrogel wherein a target RNA is tethered to the hydrogel and a ligated probe is hybridized to the target RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1208). In some embodiments, the methodny-2785575202412020440 further comprises releasing the target and / or the ligated probe (e.g., with RNase H treatment) for capture on the array (1209). In some embodiments, the method further comprises elongating the probe captured on array and proceeding with library preparation (1210).

[0321] In some embodiments, provided herein is a method for analyzing a biological sample disclosed herein may comprise embedding the biological sample, such as an FFPE sample, in a hydrogel matrix, wherein a target RNA or a ligation product may be tethered to the hydrogel matrix. In some embodiments, the target RNA or ligation product may be tethered by 3’ or 5’ tethering to the hydrogel. In some embodiments, the method may comprise removing the embedded biological sample from the hydrogel matrix as depicted in FIG. 12A, leaving the target RNA or ligation product tethered to the hydrogel matrix. In some embodiments, the removing comprises clearing the sample embedded in the hydrogel.

[0322] In some embodiments, the method as depicted in FIGS. 12A-12C may comprise applying a first probe and a second probe to the hydrogel matrix, wherein the first probe comprises a functional sequence and a sequence substantially complementary to a first portion of the target RNA. In some embodiments, the second probe may comprise a sequence substantially complementary to a second portion of the target RNA and a capture sequence configured to hybridize to or be ligated to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode. In some embodiments, the capture sequence in the second probe is substantially complementary to the capture domain in the capture probe. In some embodiments, the capture sequence comprises a sequence that is substantially complementary to a first portion of a splint oligonucleotide, which splint oligonucleotide further comprises a second portion that is substantially complementary to a sequence of the capture domain. As such, the first capture sequence and the capture domain, upon hybridization to the splint oligonucleotide, are configured to be ligated to each other, with or without gap filling prior to the ligation.

[0323] Exemplary splint oligonucleotides and methods of using splint oligonucleotides for capturing target molecules (e.g., target RNA molecules, transposed DNA, and / or analyte capture agents for proteins described in Section II-C through Section II-F of the present disclosure) to capture probes on an array include but are not limited to those described in US 2023 / 0175045 A1 and US 2023 / 0287481 A1, which are incorporated herein by reference in their entireties.ny-2785575202412020440

[0324] In some embodiments, the probe with a sequence substantially complementary to the first portion of the target RNA is referred to as a left-hand side (e.g., LHS) probe as depicted in FIG.12B. In some embodiments, the functional sequence of the first probe may comprise a functional sequence (e.g., 5’ Read 2S) as depicted in FIG. 12B. In some embodiments, the probe with a sequence substantially complementary to a second portion of the target RNA is referred to as a right-hand side (e.g., RHS) probe. In some embodiments, the capture sequence of the second probe may comprise a poly(A) sequence. As depicted in FIG. 12B, in some embodiments, the method may comprise hybridizing the first probe and the second probe to the target RNA tethered to the hydrogel matrix. In some embodiments, the method may further comprise washing the hydrogel matrix containing the tethered target RNA and the first and second probes hybridized thereon (“washes in gel” in the probe hybridization step), for instance, to remove nonspecifically hybridized first and / second probes and / or to remove first and / second probes that contain one or more mismatches to the target RNA. In some embodiments, the method further comprises ligating together the first probe and the second probe hybridized to the target RNA tethered to the hydrogel matrix, thereby generating a ligation product that is a proxy of the target RNA. In some embodiments, the method further comprises washing the hydrogel matrix containing the tethered target RNA and the ligation product hybridized thereon (“washes in gel” in the probe ligation step), for instance, to remove first and / second probes that are not ligated to each other, and / or to remove ligation products that contain one or more mismatches which are less stably hybridized to the target RNA compared to ligation products containing no mismatch with the target RNA.

[0325] In some embodiments, the first and second probes (e.g., the LHS and RHS probes shown in FIG. 12B) are provided in a probe mix which is applied to the hydrogel matrix, and the first and second probes are not immobilized to a substrate. In some embodiments, the first and second probes are dissolved in a solution such that the probes are free to move in the solution and are not immobilized to any location in the hydrogel or on the sample substrate until probe hybridization to the target RNA.

[0326] In some embodiments, the method may comprise releasing the ligation product from the hydrogel matrix. In some embodiments, the ligation product is released from the target RNA. In some embodiments, the target RNA and / or a linkage between the target RNA and the matrix is cleaved to release the tethered target RNA or a portion thereof. In someny-2785575202412020440 embodiments, the tethered target RNA is relased via cleavage of a linker between the target RNA and the hydrogel matrix. In some embodiments, the target RNA and the ligation product hybridized thereon are released together from the hydrogel matrix. In some embodiments, the ligation product is separated from the target RNA hybridized thereon, e.g., through dehybridization and / or RNA digestion, e.g., using an RNase H to cleave the phosphodiester bonds of RNA in a double-stranded RNA:DNA hybrid.

[0327] In some embodiments, the method may comprise releasing the ligation product from the hydrogel matrix and hybridizing or ligating the released ligation product to the capture domain of the capture probe on a spatial array. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate. In some embodiments, the method may comprise “sandwiching” (e.g., “Hydrogel / hydrogel” sandwich as depicted in FIG. 12A), where the RNA- tethered “tissue” hydrogel is aligned with a barcoded solid support (e.g., a spatial array such as a hydrogel barcoded capture array), for example, using a device described in any of FIGS. 1A-4B and described in Section I. In some embodiments, the method may comprise determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the ligation product or a complement thereof, and the sequence of the spatial barcode or a complement thereof.

[0328] In some embodiments, the biological sample may be a fresh cell or tissue sample. In some embodiments, the biological sample may be a frozen cell or tissue sample. In some embodiments, the biological sample may be a fixed cell or tissue sample. In some embodiments, the biological sample may be an FFPE tissue section. In some embodiments, the biological sample may be a crosslinked biological sample, and the crosslinked biological sample may be embedded in the hydrogel matrix and the target RNA in the crosslinked biological sample may be tethered to the hydrogel matrix. In some embodiments, the method comprises decrosslinking the crosslinked biological sample (e.g., an FFPE sample that has been dewaxed, rehydrated, stained (e.g., using H&E stain), and / or destained) embedded in the hydrogel matrix.

[0329] In some embodiments, the functional sequence in the first probe is in a 5’ overhang, which is not complementary to the target RNA, upon hybridization of the first probe to the target RNA. In some embodiments, the functional sequence in the first probe comprises any-2785575202412020440 primer binding sequence or a complement thereof. In some embodiments, the capture sequence in the second probe is in a 3’ overhang, which is not complementary to the target RNA, upon hybridization of the second probe to the target RNA. In some embodiments, the capture sequence in the second probe comprises a 3’ polyadenine sequence. In some embodiments, the first probe and the second probe are ligated using the target RNA as a template without gap filling prior to the ligation. In some embodiments, the first probe and the second probe are ligated with gap filling prior to the ligation. In some embodiments, the gap filling comprises hybridization of a gap-fill oligonucleotide to the target RNA and / or primer extension of the first probe or the second probe by a polymerase. In some embodiments, the gap filling comprises primer extension of the 3’end of the first probe by a polymerase with little or no strand displacement activity, thereby forming a nick between the 3’ end of the extended first probe and the 5’ end of the second probe, followed by RNA-templated ligation of the juxtaposed ends. In some embodiments, the gap filling comprises extension via a polymerase of the LHS 3’ probe end to meet the 5’ end of the RHS probe, followed by ligation. In some embodiments, hybridization of a third oligonucleotide in the gap between the first and the second probes followed by ligation of the third oligonucleotide to the LHS and the RHS probes.

[0330] In some embodiments, the first probe and / or the second probe is comprised primarily of DNA and comprises one or more ribonucleotide residues. In some embodiments, the first probe and / or the second probe comprises no more than four consecutive ribonucleotide residues. In some embodiments, the one or more ribonucleotide residues are no more than five nucleotides from the 3’ or the 5’ end of the first probe and / or the second probe. In some embodiments, the first probe comprises a 3’ terminal ribonucleotide residue. In some embodiments, the second probe comprises a 5’ flap which is cleaved (e.g., by a flap endonuclease such as FEN1) prior to ligation of the cleaved second probe to the first probe. More disclosure on flap endonuclease usage can be found, for example, in US 2020 / 0224244, which is herein incorporated by reference in its entirety.

[0331] In some embodiments, the releasing comprises releasing the tethered RNA from the hydrogel matrix. In some embodiments, the releasing comprises releasing the ligation product from the target RNA without cleaving the ligation product. In some embodiments, the releasing comprises contacting the hydrogel matrix with an RNase H.ny-2785575202412020440

[0332] In some embodiments, the capture domain in the capture probe comprises a 3’ poly(dT) sequence. In some embodiments, the method comprises generating a spatially labeled polynucleotide comprising, from 5’ to 3’, a sequence of the released ligation product and a complementary sequence of the spatial barcode. For instance, as depicted in FIG. 12C, the spatially labeled polynucleotide can be generated by primer extension of the poly(A) hybridized to the poly(dT)VN using the capture probe as a template, and the spatially labeled polynucleotide comprises, from 5’ to 3’: a functional sequence (e.g., 5’ Read 2S), a sequence of the first probe, a sequence of the second probe, poly(A), a complement of the UMI, a complement of the spatial barcode, and a complement of a functional sequence in the capture probe (e.g., Read 1). In some embodiments, the method comprises generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released ligation product.

[0333] In some embodiments, the spatially labeled polynucleotide or a portion thereof may be released from the array for analysis. In some embodiments, the spatially labeled polynucleotide or portion thereof released from the array may be analyzed by nucleic acid sequencing. In some embodiments, an amplification (e.g., a linear amplification) can be performed on-surface and post-extension to release a spatially labeled polynucleotides for sequencing analysis. In some embodiments, after extension of a captured ligation product of probes ligated using an RNA as a template, extended products (e.g., single copies) can be denatured and amplified, e.g., in a tube. In some embodiments, after extension of a captured ligation product of probes ligated using an RNA as a template, extended products can be treated with an enzyme (e.g., Exo I selectively catalyzes the degradation of single-stranded DNA in the 3′ to 5′ direction, whereas double-stranded DNA is protected from degradation) and amplified, e.g., using linear amplification after extension, and the amplification products (several copies per molecule) in the supernatant of the reaction can be collected. Variation of this approach can be implemented in any of the embodiments herein, including embodiments involving reverse transcription of a 3’ or 5’ tethered RNA and embodiments involving RNA-templated ligation of probes on a 3’ or 5’ tethered RNA.

[0334] An overview for spatial transcriptomic profiling of FFPE tissue sections mediated by tethering of the 3’ end of a target mRNA molecule to a hydrogel via a hydrogel cleavable linker in accordance with some embodiments is depicted in FIG. 12A. In someny-2785575202412020440 embodiments, an FFPE tissue section may be provided on a slide. In some embodiments, the tissue section may be baked, dewaxed and / or rehydrated. In some embodiments, an H&E stain may be applied and / or the tissue sample may be imaged, optionally using bright field microscopy (e.g., BF imaging). In some embodiments, the biological sample may be destained. The hydrogel (e.g., hydrogel mesh or hydrogel matrix) formation step may be carried out to intermesh (e.g., embed) the biological sample. During the formation of the sample intermeshed hydrogel (e.g., sample hydrogel), RNA molecules may be 3’ tethered to the hydrogel matrix by a cleavable linker. During the formation of the sample intermeshed hydrogel (e.g., sample hydrogel), RNA molecules may be 5’ tethered to the hydrogel matrix by a cleavable linker. In some embodiments, the cleavable linker comprises a cleavable segment. In some embodiments, the cleavable segment may comprise a disulfide bond. The tissue may be decrosslinked after tethering of the target molecule (e.g., target RNA or target nucleic acid) to the hydrogel matrix. In some embodiments, the tissue sample or components of the tissue sample may be removed by a clearing process leaving the RNA tethered (e.g., target molecule) to the hydrogel matrix. An RNA templated ligation probe (e.g., RTL probe) hybridization may be performed on the RNA tethered to the hydrogel. In the first step of RTL probe hybridization, in accordance with some embodiments, two probes including a left-hand side (LHS) probe and a right-hand side (RHS) probe may be hybridized to an mRNA target site of the tethered RNA as depicted in FIG. 12B. The LHS probe may include, in part, a Read 2S segment starting at its 5’ end. The RHS probe may include, in part, a poly(A) segment ending at its 3’ end as depicted in FIG. 12B. In some embodiments, following the probe hybridization step may be a probe ligation step. In some embodiments, the slide holding the ligated RNA product tethered to the hydrogel may be sandwiched to a hydrogel barcoded capture array. In some embodiments, capture probes (e.g., capture agents) of the hydrogel barcoded capture array may be immobilized to a second hydrogel intermeshed (e.g., second hydrogel mesh) with the capture probe of the barcoded capture array. In some embodiments, each capture probe may comprise a linker and an oligonucleotide sequence comprising a Read 1 sequence, a spatial barcode sequence, a UMI sequence, a poly(dT)VN sequence, or any combination of, as depicted in FIG. 12C. In some examples, the capture probe may capture the RNA target by hybridization of the poly(dT)VN sequence to the poly(A) section of the ligation product as depicted in FIGS. 12A and 12C. Following capture, inny-2785575202412020440 accordance with some embodiments, the probe elongation and / or library preparation steps may be performed.

[0335] In some embodiments, provided herein is a method for determining a spatial transcriptomic profile for a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target RNA from the biological sample is tethered to a matrix of the hydrogel; b) digesting and removing the embedded biological sample from the hydrogel; c) releasing the target RNA from the matrix of the hydrogel; d) hybridizing the released target RNA to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode; e) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the target RNA is tethered to the hydrogel matrix via a functional group at a 3’ end of the target RNA. In some embodiments, the target RNA is tethered to the hydrogel matrix via a functional group at a 5’ end of the target RNA.

[0336] Turning to FIG. 13A, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. a fresh- frozen tissue sample) with the steps of 1) H&E staining the tissue sample; 2) imaging the tissue sample (e.g., using brightfield (BF)); and / or 3) destaining the tissue sample (1301). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1302). In some embodiments, the method further comprises tethering a target RNA (e.g., a target mRNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1303). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1304). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared tissue sample (e.g., a “tissue” hydrogel wherein a target RNA is tethered to the hydrogel and a ligated probe is bound to the target RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1305). In some embodiments, the method further comprises releasing the target RNA (e.g., by cleaving cleavable linkers or by changing the pH of a boronic acid-based hydrogel) for capture on the array (e.g., hydrogel barcoded capture array) (1306). In some embodiments, the method further comprises 1) reverse transcribing the target RNA captured on the array (e.g.,ny-2785575202412020440 hydrogel barcoded capture array) and 2) performing template switching and second-strand synthesis (1307) to generate a spatially labeled polynucleotide disclosed herein. In some embodiments, the method further comprises 1) performing amplification of a spatially labeled polynucleotide, e.g., amplification of the second strand synthesis product, and 2) proceeding with library preparation (1308).

[0337] In some embodiments, provided herein is a method that comprises embedding the biological sample in a hydrogel matrix, wherein a target RNA may be tethered to the hydrogel matrix; removing the embedded biological sample from the hydrogel matrix; releasing the target RNA from the hydrogel matrix; d) hybridizing or ligating the released target RNA to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof. In some embodiments, the target RNA may be 3’ tethered to the hydrogel matrix. In some embodiments, the target RNA may be 5’ tethered to the hydrogel matrix. In some embodiments, the capture domain in the capture probe comprises a 3’ poly(dT) sequence as depicted in FIG. 13B, and the poly(A) tail of the released target RNA hybridizes to the 3’ poly(dT) sequence. In some embodiments, the method comprises extending the capture probe by a polymerase (e.g., a reverse transcriptase) using the captured target RNA as a template, thereby generating an extended capture probe comprising, from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released target RNA as depicted in FIG. 13B. In some embodiments, the extended capture probe as depicted in FIG. 13B may further comprise a 3’ end homopolymer sequence, such as a poly(C) sequence. In some embodiments, the method may further comprise hybridizing a template switch oligonucleotide to the extended capture probe (e.g., via hybridization of rGrGrG to the CCC sequence in the extended capture probe, as shown in FIG. 13C), and extending the extended capture probe using the template switch oligonucleotide as a template to generate a further extended capture probe. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate.ny-2785575202412020440

[0338] As depicted in FIG. 13C, the method may further comprise hybridizing a primer (“Second Strand Primer” as shown in the figure) to the further extended capture probe at a sequence complementary to the template switch oligonucleotide (TSO), and extending the primer using the further extended capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a sequence of the released target RNA and a complementary sequence of the spatial barcode as depicted in FIG. 13C. In some embodiments, the spatially labeled polynucleotide or a portion thereof may be released from the further extended capture probe for analysis. In some embodiments, the spatially labeled polynucleotide or portion thereof released from the array may be analyzed by nucleic acid sequencing. In some embodiments, after second strand-synthesis an Exo I treatment can be performed to get rid of the unused capture probes, followed by amplification. Exo I treatment prevents undesired priming of extended spatially labeled polynucleotides with proximal capture probes available on the surface of the array during amplification.

[0339] In some embodiments, provided herein is a method for determining a spatial transcriptomic profile for a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target RNA is tethered to a matrix of the hydrogel matrix; b) removing the embedded biological sample from the hydrogel; c) hybridizing a probe that is substantially complementary to a portion of the target RNA tethered to the matrix of the hydrogel; d) extending the hybridized probe wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe; e) releasing the extended probe from the hydrogel; f) capturing the extended probe to a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode; and g) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0340] Turning to FIG. 14A, a method using a TSO-based capture approach for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g., a fresh-frozen tissue sample) with the steps of 1) H&E staining the tissue sample; 2) imaging the tissue sample (e.g., using brightfield (BF)); and / or 3) destaining the tissue sample (1401). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1402). In some embodiments, the method furtherny-2785575202412020440 comprises tethering a target RNA (e.g., a target mRNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1403). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1404). In some embodiments, the method further comprises reverse transcribing the tethered RNA within matrix (e.g. while tethered to hydrogel) (1405). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared tissue sample (e.g., a “tissue” hydrogel where a target RNA is tethered to the hydrogel and cDNA derived from the tethered RNA is bound to the tethered RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1406). In some embodiments, the method further comprises 1) releasing the cDNA and / or the target RNA from the matrix by a) reversing linker or dissolving matrix, or b) digesting target RNA tethered to matrix (e.g. using RNase H), and 2) capturing released cDNA and / or target RNA on an array such as a hydrogel barcoded capture array (e.g., using poly(rG) as a capture sequence) (1407). In some embodiments, the method further comprises performing on the captured cDNA and / or target RNA template switching and transcript extension using template switch oligo (TSO) priming (1408). In some embodiments, the method further comprises 1) performing amplification of the spatially labeled polynucleotide, e.g., a product of the captured target RNA and / or a product of the cDNA, and / or 2) proceeding with library preparation (1409).

[0341] In some embodiments, provided herein is a method that comprises: embedding the biological sample in a hydrogel matrix, wherein a target RNA may be tethered to the hydrogel matrix; removing the embedded biological sample from the hydrogel matrix; hybridizing a probe that may be substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; extending the hybridized probe wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe and releasing the extended probe from the hydrogel matrix. In some embodiments, the method further comprises hybridizing or ligating the extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof as depicted in FIG. 14. In some embodiments, the probe hybridizes to a poly(A) sequence in theny-2785575202412020440 target RNA tethered to the hydrogel matrix. In some embodiments, the probe comprises a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence as depicted in FIG. 14. In some embodiments, the capture domain in the capture probe comprises a template switch oligonucleotide sequence and a homopolymer sequence (e.g., rGrGrG) complementary to the homopolymer sequence (e.g., CCC) at the 3’ end of the extended probe as depicted in FIG. 14. In some embodiments, the method may further comprise extending the extended probe using the capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a complementary sequence of the released target RNA and a complementary sequence of the spatial barcode as depicted in FIG. 14. In some embodiments, the spatially labeled polynucleotide or a portion thereof may be released from the capture probe for analysis. In some embodiments, the spatially labeled polynucleotide or portion thereof released from the spatial array may be analyzed by nucleic acid sequencing. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate.

[0342] In some examples, provided herein are methods for the spatial transcriptomic profiling of fresh frozen (e.g., FF) tissue sections mediated by 5’ target tethering to a hydrogel, utilizing a 5’ RT-based gene expression analysis and TSO-based capture. In some embodiments, an FF tissue section may be provided on a slide. In some embodiments, the tissue section may be H&E stained, imaged with a bright field configured microscope and / or destained. In some embodiments, a hydrogel (e.g., hydrogel matrix) may be formed within the tissue section. RNA target molecules (e.g., target RNA, target nucleic acid) may be tethered to the hydrogel matrix by a cleavable linker. In some examples, the cleavable linker may comprise a cleavable segment. In some examples, the cleavable segment may comprise a disulfide bond. In some examples, a clearing step (e.g., tissue removal) may be performed leaving target molecules tethered to the hydrogel. In some examples, following clearing, a reverse transcription step may be performed on the biological sample intermeshed with the sample gel to reverse transcribe the tethered RNA target molecule. In some embodiments, a sequence containing poly(dT) and non-poly(dT) sequences may be extended by reverse transcription. The reverse transcription product is then captured by the capture probe immobilized to the spatial array to generate a spatially labeled polynucleotide. Sequencing of the spatially labeled polynucleotide or a complement thereofny-2785575202412020440 allows for association of the spatial barcode to the target molecule at a location in the biological sample, thereby revealing a spatial profile of the target molecule in the biological sample.

[0343] In some embodiments, provided herein is a method for determining a spatial transcriptomic profile for a biological sample, the method comprising: a) embedding the biological sample in a hydrogel, wherein a target RNA from the biological sample is tethered to a matrix of the hydrogel; b) removing the embedded biological sample from the hydrogel; c) hybridizing a first probe that is substantially complementary to a portion of the target RNA tethered to the matrix of the hydrogel; d) extending the hybridized first probe wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe; e) hybridizing a second probe to the extended probe in the hydrogel and further extending the extended probe using the second probe as a template, wherein the further extension comprises incorporating a polyuridine-polyadenine double stranded region at the end of a duplex formed between the second probe and the further extended probe; c) releasing the further extended probe from the hydrogel; d) capturing the further extended probe by a capture domain of a capture probe on an array, wherein the capture probe further comprises a spatial barcode; e) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the further extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

[0344] Turning to FIG. 15, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. a fresh- frozen tissue sample) with the steps of 1) H&E staining the tissue sample; 2) imaging the tissue sample (e.g., using brightfield (BF)); and / or 3) destaining the tissue sample (1501). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1502). In some embodiments, the method further comprises tethering a target RNA (e.g., a target mRNA) in the sample to the matrix (e.g., through 3’ tethering of target nucleic acid via an optional linker) (1503). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1504). In some embodiments, the method further comprises reverse transcribing the tethered RNA within matrix (e.g. while tethered to hydrogel) (1505). In some embodiments, the method further comprises transcript switching oligo (TSO) priming. In some embodiments, the method further comprises contacting a transcript switching oligo (TSO) primer to the resulting cDNA within the matrixny-2785575202412020440 (1506). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared tissue sample (e.g., a “tissue” hydrogel wherein a target RNA is tethered to the hydrogel and cDNA is bound to the target RNA) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1507). In some embodiments, the method further comprises releasing the cDNA and / or target RNA by digesting the target RNA tethered to matrix (e.g. using RNase H) (1508). In some embodiments, the method further comprises performing second strand synthesis on the cDNA hybridized to the array (e.g. the matrix barcoded capture array) (1509). In some embodiments, the method further comprises performing amplification of a spatially labeled polynucleotide, e.g., amplification of the product of second strand synthesis of the captured cDNA, and 2) proceeding with library preparation (1510).

[0345] In some embodiments, provided herein is a method that comprises: embedding the biological sample in a hydrogel matrix, wherein a target RNA from the biological sample may be tethered to the hydrogel matrix; removing the embedded biological sample from the hydrogel matrix; hybridizing a first probe that may be substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; extending the hybridized probe wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe, and hybridizing a second probe to the extended probe in the hydrogel matrix and further extending the extended probe using the second probe as a template, wherein the extension comprises incorporating a polyuridine-polyadenine double stranded region at the end of a duplex formed between the second probe and the further extended probe. In some embodiments, the method may further comprise releasing the further extended probe from the hydrogel matrix. In some embodiments, the released further extended probe comprises the double stranded region or a portion thereof. In some embodiments, the released further extended probe is single stranded, e.g., when RNase H is used to digest the RNA strand (which is tethered to the hydrogel matrix) in the duplex which comprises a RNA:DNA hybrid, the DNA strand can be released from the hydrogel matrix as a single stranded further extended probe. In some embodiments, the method may further comprise hybridizing or ligating the further extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode. In some embodiments, the method may further comprise determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the furtherny-2785575202412020440 extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, as depicted in FIG. 15. In some embodiments, the first probe hybridizes to a poly(A) sequence in the target RNA tethered to the hydrogel matrix as depicted in FIG. 15. In some embodiments, the first probe comprises a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence. In some embodiments, the second probe comprises a 5’ poly(U) sequence, a template switch oligonucleotide sequence, and a homopolymer sequence (e.g., rGrGrG) complementary to the homopolymer sequence (e.g., CCC) at the 3’ end of the extended probe. In some embodiments, the homopolymer sequence comprises poly(rG). In some embodiments, the further extended probe comprises a 3’ poly(A) sequence, which is generated using the 5’ poly(U) sequence as a template. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate.

[0346] In some embodiments, the releasing comprises releasing the tethered RNA from the hydrogel matrix. In some embodiments, the releasing comprises releasing the further extended probe from the target RNA. In some embodiments, the releasing comprises contacting the hydrogel matrix with an RNase H as depicted in FIG. 15. In some embodiments, the capture domain in the capture probe comprises a 3’ poly(dT) sequence. In some embodiments, the method comprises generating a spatially labeled polynucleotide comprising: i) from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released further extended probe, or ii) from 5’ to 3’: a sequence of the released further extended probe and a complementary sequence of the spatial barcode. In some embodiments, the spatially labeled polynucleotide or a portion thereof may be released from the array for analysis. In some embodiments, the spatially labeled polynucleotide or portion thereof released from the array may be analyzed by nucleic acid sequencing. D. Spatial Profiling of Open Chromatin, Protein Binding to Chromatin, and Chromatin Modifications

[0347] A method disclosed herein can be used not only in gene expression profiling (e.g., as described in Section II-C), but also in spatial chromatin profiling, such as spatial ATAC which combines the assay for transposase-accessible chromatin and sequencing (ATAC-seq). The gene expression profiling and spatial ATAC-seq can be performed separately (e.g., on 109ny-2785575202412020440 separate biological samples or in separate regions of interest in the same biological sample), or the gene expression profiling and spatial ATAC-seq can be combined in one assay run performed on the same biological sample or the same region of interest thereof.

[0348] In some embodiments, disclosed herein is a method for profiling open chromatin in a biological sample, comprising: a) contacting the biological sample with a plurality of transposomes, wherein a transposome of the plurality comprises (i) a capture sequence that is complementary to a capture domain of a capture probe on a spatial array, (ii) a linker sequence, and (iii) a reactive group; b) tagmenting the open chromatin with the plurality of transposomes; c) embedding the biological sample in a hydrogel and tethering the tagmented open chromatin to the hydrogel via the reactive group of the tagmented open chromatin; d) removing the biological sample from the hydrogel; e) releasing the tagmented open chromatin from the hydrogel and hybridizing the released tagmented open chromatin to the capture domain of the capture probe on the spatial array, wherein the capture probe further comprises a spatial barcode; and f) determining the profile of the open chromatin in the biological sample by determining (i) the sequence of all or a portion of the tagmented open chromatin, or a complement thereof, and (ii) the sequence of all or a portion of the spatial barcode, or a complement thereof. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and no hydrogel on the array substrate.

[0349] Turning to FIGS. 16A-16F, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. a fresh-frozen tissue sample) with the steps of 1) fixation of the tissue sample; 2) H&E or immunofluorescence (IF) staining of the tissue sample; 3) imaging the tissue sample (e.g., using brightfield (BF) or fluorescence imaging); 4) destaining the tissue sample; and / or 5) permeabilization of the tissue sample (e.g. gentle permeabilization) (1601). In some embodiments, the method further comprises preparing and / or activating RNA targets within the tissue sample for 3’ or 5’ tethering (e.g. “polishing” ends of RNA for attachment to boronic acid moieties) (1602). In some embodiments, the method further comprises performing transposition on targets (e.g., genomic DNA) within tissue sample (e.g., ‘tagmentation’ using Tn5) (1603). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., any-2785575202412020440 hydrogel) (1604). In some embodiments, the method further comprises tethering a target RNA (e.g., a target mRNA) in the sample to the matrix (e.g., through either 5’ or 3’ tethering of target nucleic acid via an optional linker) (1605). In some embodiments, the method further comprises decrosslinking the tissue sample from the matrix (1606). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1607). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared sample (e.g., a “tissue” hydrogel where a target RNA is tethered to the hydrogel) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1608). In some embodiments, the method further comprises releasing the target RNA tethered to matrix (e.g. using RNase H) and capturing on array (e.g., hydrogel barcoded capture array) (1609).

[0350] In some embodiments, provided herein is a method wherein a biological sample is contacted with a plurality of transposomes, wherein a transposome of the plurality comprises (i) a capture sequence that may be complementary to a capture domain of a capture probe on the array, (ii) a linker, and (iii) a reactive group, thereby tagmenting an open chromatin in the biological sample with the plurality of transposomes, and wherein the tagmented open chromatin may be tethered to the hydrogel matrix via the reactive group of the tagmented open chromatin, wherein after the biological sample may be removed from the hydrogel matrix, the tagmented open chromatin may be released from the hydrogel matrix, the released tagmented open chromatin may be hybridized or ligated to a capture domain of a capture probe on the array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the tagmented open chromatin or a complement thereof and (ii) the spatial barcode or a complement thereof may be determined.

[0351] An overview of the method for analyzing a biological sample, in accordance with some embodiments, may comprise spatial ATAC and 3’ or 5’-gene expression workflow as depicted in FIG. 16A. In some embodiments, the workflow may comprise the steps of: fixing the FF tissue section; H&E / IF staining; imaging, destaining; permeabilizing the biological sample; preparing and activating the RNA target for 3’ or 5’ tethering; tagmentation (e.g., using a Tn5 transposome); hydrogel matrix embedding (during and / or after which the target RNA molecules and transposed DNA can be tethered to the hydrogel matrix); descrosslinking the tissue in the hydrogel matrix; removing the tissue (e.g., by clearing), while the target molecules (e.g., target 111ny-2785575202412020440 RNA molecules and transposed DNA) remain tethered to the hydrogel matrix; and simultaneously releasing the target molecules (e.g., target RNA molecules and transposed DNA) from the sample hydrogel matrix and capturing the released molecules with the hydrogel barcoded capture array. In some embodiments, target RNA molecules for the RNA-seq modality and transposed DNA for the ATAC-seq modality are captured by different capture probes on the hydrogel barcoded capture array, for instance, as shown in FIG. 19.

[0352] In some embodiments, the permeabilization step may be carried out gently to facilitate the RNA target preparation step. In some embodiments, after the clearing step the target molecules (e.g., target RNA molecules and transposed DNA) remain in the hydrogel. The target releasing step may comprise contacting the sample hydrogel (e.g., tissue hydrogel, substrate or first substrate) with the hydrogel barcoded capture array (e.g., second substrate). The contacting step may also be referred to as the sandwiching step. Tethered target molecules (e.g., target RNA molecules and transposed DNA) may be released by cleaving a cleavable linker. The cleavable linker may comprise a disulfide bond that may be chemically cleaved. In some embodiments, following release and capture of target molecules (e.g., target RNA molecules and transposed DNA), workflow may proceed with a step of amplifying material and / or generating libraries. Subsequent analysis may then be performed, which may in some instances include associating the spatial barcode with locations in the biological sample, thereby identifying the presence of the target molecules (e.g., target RNA molecules and transposed DNA) at locations in the biological sample.

[0353] FIGS. 16B-16D depict an exemplary transposition step where, in certain circumstances a transposome complex, in this example a transposome comprising a Tn5 transposase and modified transposon ends, may be modified for tethering of transposed DNA. A modified design for a transposon end in the transposome complex, in accordance with some embodiments, is depicted in FIG. 16B. In certain aspects, the transposon end (e.g., mosaic end) may comprise a PCR handle (pR2), a cleavable (e.g., disulfide or photo-cleavable) linker, and a 5’-acrydite at its terminus. The acrydite functionality may be a reactive group for hydrogel embedding. In some embodiments, the disulfide linker may allow for chemically triggered release (e.g., by treatment of a reducing agent such as dithiothreitol (DTT)) of the tethered target molecule (e.g., the transposed DNA). Assembly of a partial capture sequence and Tn5 mosaic end duplex with the PCR handle (pR2) and Tn5 transposase, in accordance with someny-2785575202412020440 embodiments, is depicted in FIG. 16C. The structure of the transposed DNA, in accordance with some embodiments, is depicted in FIG. 16D. In some embodiments, the acrydite group allows for tethering of a transposed DNA (e.g., from an open chromatin region) to the sample hydrogel. Any transpositional system can be equally applicable, for example a Mu, Vibrio, Marinar, Tn7, or other transpositional system once modified could also be used to tagment the accessible chromatin and used in tethering methods as described herein.

[0354] The structures of capture probes on the hydrogel barcoded array, in accordance with some embodiments, are depicted in FIGS. 16E-16F. For example, one of the capture domains may be specific to capturing RNA or an RNA proxy using a poly(T) capture domain. The second capture domain may be specific to a mosaic end of a transposon, such that tagmented accessible DNA is captured concurrently by those specific capture domains of capture probes on an array. As such, more than one target nucleic acid can be captured concurrently on the sample array depending on the capture domain sequences on the array, thereby determining the spatial expression patterns of, in this example, RNA or a proxy thereof as well as accessible chromatin from a sample. FIG. 7 provides examples of different capture domains that could be used for different target nucleic acid capture from a biological sample, such as using a homopolymer capture domain, a fixed sequence capture domain, or a degenerate or partially degenerate capture domain.

[0355] In some embodiments, a molecule comprising transposed DNA of the ATAC- seq modality can be captured (via the partial capture sequence) by the capture probe comprising the spacer shown in FIG. 16E. In one example, the spacer in the capture probe can comprise a sequence substantially complementary to a sequence in the partial capture sequence of the transposed DNA, such that the spacer can hybridize to the partial capture sequence, followed by ligation with or without gap filling prior to the ligation. In one example, a splint oligonucleotide comprising a first sequence substantially complementary to the spacer in the capture probe and a second sequence substantially complementary to the partial capture sequence in the transposed DNA can be used to connect the capture probe to the transposed DNA by ligation using the splint oligonucleotide as a template, with or without gap filling prior to the ligation. A molecule comprising a transcript sequence or complement thereof of the RNA-seq modality can be captured (via the poly(A) sequence) by the capture probe comprising a poly(dT) sequence shown in FIG. 16F.ny-2785575202412020440

[0356] Turning to FIGS. 17A-17D, a method for analyzing a biological sample is disclosed herein. In some embodiments, the method comprises preparing a tissue sample (e.g. a fresh-frozen tissue sample) with the steps of 1) fixation of the tissue sample; 2) H&E or immunofluorescence (IF) staining of the tissue sample; 3) imaging the tissue sample (e.g., using brightfield (BF) or fluorescence imaging); 4) destaining the tissue sample; and / or 5) permeabilization of the tissue sample (e.g. gentle permeabilization) (1701). In some embodiments, the method further comprises preparing and / or activating RNA targets within the tissue sample for 3’ or 5’ tethering (e.g. “polishing” ends of RNA for attachment to boronic acid moieties) (1702). In some embodiments, the method further comprises performing primary and secondary antibody incubations on the tissue sample (1703). In some embodiments, the method further comprises performing transposition on tissue sample (e.g. using pA-Tn5) (1704). In some embodiments, the method further comprises embedding the sample in a matrix (e.g., a hydrogel) (1705). In some embodiments, the method further comprises decrosslinking the tissue sample from the matrix (1706). In some embodiments, the method further comprises clearing sample (e.g., tissue removal) while the tethered target remains attached to matrix (1707). In some embodiments, the method further comprises sandwiching the matrix-embedded and cleared sample (e.g., a “tissue” hydrogel where a target RNA is tethered to the hydrogel) between a substrate (e.g., slide) with an array (e.g., barcoded capture array embedded in another matrix, such as a hydrogel barcoded capture array) (1708). In some embodiments, the method further comprises releasing the target RNA tethered to matrix (e.g. by changing the pH of a boronic acid-based hydrogel) and capturing the target RNA on the array (e.g., hydrogel barcoded capture array) (1709).

[0357] In some embodiments, provided herein is a method wherein a biological sample, in accordance with some embodiments, is contacted with an antibody that binds to a chromatin protein or chromatin-associated protein in the biological sample, and with a transposome-binding moiety complex that may bind to the antibody, wherein the transposome- binding moiety complex may comprise (i) a transposase (e.g., Tn5), (ii) an antibody-binding moiety (e.g., protein A), (iii) a transposon end sequence comprising a capture sequence that may be complementary to a capture domain of a capture probe on a spatial array, (iv) a reactive group, or a combination thereof, thereby tagmenting a genomic DNA in the biological sample with the aide of the transposome-binding moiety complex. In some embodiments, theny-2785575202412020440 transposome-binding moiety complex comprises a transposone and a binding moiety. In such embodiments, the tagmented genomic DNA may be tethered to the hydrogel matrix via the reactive group of the transposome-binding moiety complex, wherein after the biological sample may be removed from the hydrogel matrix, the tagmented genomic DNA may be released from the hydrogel matrix, the released tagmented genomic DNA may be hybridized or ligated to a capture domain of a capture probe on the spatial array. In some embodiments, the capture probe may further comprise a spatial barcode. In some embodiments, the spatial array comprises capture probes comprising spatial barcodes and a hydrogel on the array substrate. In some embodiments, the spatial array comprises capture ...

Claims

202412020440 CLAIMS 1. A method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a matrix; b) tethering a target nucleic acid in the biological sample to the matrix; c) removing a subset of cellular components from the biological sample in the matrix; d) releasing the target nucleic acid from the matrix; e) hybridizing or ligating the released target nucleic acid to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and f) determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

2. The method of claim 1, wherein the target nucleic acid comprises DNA and / or RNA.

3. The method of claim 2, wherein the RNA is mRNA.

4. The method of any one of claims 1-3, wherein the matrix is a hydrogel matrix.

5. The method of any one of claims 1-4, wherein the tethering comprises tethering a 3’ end of the target nucleic acid to the matrix.

6. The method of any one of claims 1-4, wherein the tethering comprises tethering a 5’ end of the target nucleic acid to the matrix.

7. The method of any one of claims 1-6, wherein the tethering comprises enzymatic tethering.

8. The method of any one of claims 1-7, wherein the tethering comprises non-enzymatic tethering.

9. The method of any one of claims 1-8, wherein the tethering comprises a periodate oxidation reaction or a reaction with boronic acid.

10. The method of any one of claims 1-9, wherein the tethering comprises tethering the target nucleic acid to the matrix via a linker.ny-2785575202412020440 11. The method of any one of claims 1-10, wherein the linker comprises a cleavable linker.

12. The method of claim 11, wherein the cleavable linker comprises a disulfide bond.

13. The method of any one of claims 1-12, wherein the matrix is formed using N,N′- Bis(acryloyl)cystamine (BAC) as a crosslinker.

14. The method of any one of claims 1-13, wherein the removing of the subset of cellular components from the biological sample comprises removing at least a subset of ribosomes from the biological sample.

15. The method of any one of claims 1-14, wherein the releasing comprises contacting the matrix with a chemical agent configured to cleave a cleavable linker between the target nucleic acid and the matrix.

16. The method of any one of claims 1-14, wherein the releasing comprises illuminating the matrix with light, thereby cleaving a cleavable linker between the target nucleic acid and the matrix.

17. The method of any one of claims 1-14, wherein the releasing comprises altering the pH of a solution in contact with the matrix, thereby releasing the tethered target nucleic acid.

18. The method of claim 17, wherein the target nucleic acid is tethered to the matrix via a boronate ester bond.

19. The method of claim 18, wherein the boronate ester bond is formed between a boronic acid moiety and 3’ diols of the target nucleic acid, wherein the target nucleic acid is RNA.

20. The method of any one of claims 1-19, wherein the matrix comprises a boronic acid- based hydrogel matrix.

21. The method of any one of claims 1-20, wherein the releasing comprises exposing the matrix to a temperature of about 40°C to about 75°C, thereby releasing the tethered target nucleic acid, optionally wherein the matrix is a hydrogel matrix comprising poly(N- isopropylacrylamide).

22. The method of any one of claims 1-21, wherein the target nucleic acid comprises a cellular nucleic acid in the biological sample, a product of the cellular nucleic acid generated inny-2785575202412020440 the biological sample, a nucleic acid probe that directly or indirectly binds to the cellular nucleic acid, or a product of the nucleic acid probe generated in the biological sample.

23. The method of any one of claims 1-22, wherein the matrix and the biological sample embedded therein are on a first solid support, and the spatial array is on a second solid support.

24. The method of any one of claims 1-23, wherein the sequence of all or a portion of the target nucleic acid, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof, is determined using nucleic acid sequencing.

25. The method of claim 24, wherein the nucleic acid sequencing comprises sequencing by synthesis, sequencing by ligation, sequencing by binding, sequencing by avidity, sequencing by hybridization, or any combination thereof.

26. The method of any one of claims 1-25, further comprising directing the released target nucleic acid to the capture probe.

27. The method of claim 26, wherein the directing comprises directing via a capacitor, thereby generating an electrophoretic migration of the released target nucleic acid from the matrix to the spatial array.

28. The method of claim 27, wherein at least one plate of the capacitor comprises a coating of indium tin oxide.

29. The method of any one of claims 1-28, wherein the matrix is a first hydrogel matrix and the capture probe is affixed on the spatial array and embedded in a second hydrogel matrix.

30. The method of claim 29, wherein the first hydrogel matrix and the second hydrogel matrix are of the same composition.

31. The method of claim 29, wherein the first hydrogel matrix and the second hydrogel matrix are of different compositions.

32. The method of any one of claims 1-31, comprising determining a spatial gene expression profile of the target nucleic acid in the biological sample.ny-2785575202412020440 33. The method of any one of claims 1-32, wherein a plurality of different target nucleic acids are tethered to the matrix in a), released from the matrix in c), and hybridized or ligated to capture domains of capture probes on the spatial array.

34. The method of any one of claims 1-33, comprising digesting and / or clearing the embedded biological sample from the matrix.

35. The method of any one of claims 1-34, wherein the biological sample is a fresh tissue sample, a frozen tissue sample, or a fixed tissue sample.

36. The method of any one of claims 1-35, wherein the biological sample is a fresh frozen tissue section or a formalin-fixed paraffin-embedded tissue section.

37. The method of any one of claims 1-36, comprising fixing the biological sample prior to embedding in the matrix, and decrosslinking the embedded biological sample prior to or during the removing of the subset of cellular components.

38. A method for analyzing a biological sample, the method comprising: a) contacting a biological sample comprising a target nucleic acid with an attachment agent comprising a boronic acid moiety capable of covalently reacting with at least one 2’,3’ vicinal diol of the target nucleic acid and an attachment moiety capable of attaching covalently or noncovalently to a matrix-forming agent in the biological sample, wherein the biological sample and the attachment agent are contacted under conditions suitable to form a covalent bond between the boronic acid moiety and the 2’,3’ vicinal diol of the target nucleic acid; b) contacting the biological sample with the matrix-forming agent, thereby forming a matrix embedding the biological sample and tethering the target nucleic acid to the matrix; c) clearing the embedded biological sample from the matrix; d) releasing the target nucleic acid from the matrix; e) capturing the target nucleic acid by a capture probe on a spatial array, wherein the capture probe comprises a capture domain and a spatial barcode; and f) determining the sequence of all or a portion of the target nucleic acid, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof.

39. A method for analyzing a biological sample, the method comprising:ny-2785575202412020440 a) contacting the biological sample comprising a fragmented target nucleic acid with a 3’ phosphatase to provide a fragmented target nucleic acid comprising a 2’,3’-vicinal diol moiety; b) contacting the biological sample with a formylation reagent, wherein the formylation reagent converts the 2’,3’-vicinal diol moiety into a 2’,3’-dialdehyde moiety, optionally wherein the formylation reagent comprises sodium (meta)periodate; c) contacting the biological sample with an attachment agent and a matrix-forming agent, the attachment agent comprising at least one aldehyde-reactive group capable of forming a covalent bond with at least one aldehyde of the 2’,3’-dialdehyde moiety of the fragmented target nucleic acid and an attachment moiety capable of attaching covalently or noncovalently to the matrix-forming agent, thereby forming a matrix embedding the biological sample and tethering the fragmented target nucleic acid to the matrix; d) clearing the embedded biological sample from the matrix; e) releasing the fragmented target nucleic acid from the matrix; f) hybridizing the fragmented target nucleic acid to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and g) determining the sequence of all or a portion of the fragmented target nucleic acid, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof.

40. The method of any one of claims 1-39, wherein the capture probe further comprises a cleavage domain, a functional domain, a unique molecular identifier, or a combination thereof.

41. A method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) applying a first probe and a second probe to the hydrogel matrix, wherein the first probe comprises a functional sequence and a sequence substantially complementary to a first portion of the target RNA and the second probe comprises a sequence substantially complementary to a second portion of the target RNA and a capture sequence that is configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode;ny-2785575202412020440 d) hybridizing the first probe and the second probe to the target RNA tethered to the hydrogel matrix; e) ligating together the first probe and the second probe hybridized to the target RNA tethered to the hydrogel matrix, thereby generating a ligation product that is a proxy of the target RNA; f) releasing the ligation product from the hydrogel matrix and hybridizing or ligating the released ligation product to the capture domain of the capture probe on the spatial array; and g) determining a spatial transcriptomic profile for the biological sample by determining a sequence of all or a portion of the ligation product, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof.

42. The method of claim 41, wherein prior to a), the biological sample is a crosslinked biological sample, and wherein in a), the crosslinked biological sample is embedded in the hydrogel matrix and the target RNA in the crosslinked biological sample is tethered to the hydrogel matrix.

43. The method of claim 42, wherein between a) and b), the method comprises decrosslinking the crosslinked biological sample embedded in the hydrogel matrix.

44. The method of any one of claims 41-43, wherein prior to a), the biological sample is a fixed tissue sample.

45. The method of any one of claims 41-44, wherein prior to a), the biological sample is a formalin-fixed paraffin-embedded (FFPE) tissue section.

46. The method of any one of claims 41-45, wherein the target RNA is 3’ tethered to the hydrogel matrix.

47. The method of any one of claims 41-45, wherein the target RNA is 5’ tethered to the hydrogel matrix.

48. The method of any one of claims 41-47, wherein the functional sequence in the first probe is in a 5’ overhang upon hybridization of the first probe to the target RNA.

49. The method of any one of claims 41-48, wherein the functional sequence in the first probe comprises a primer binding sequence or a complement thereof.ny-2785575202412020440 50. The method of any one of claims 41-49, wherein the capture sequence in the second probe is in a 3’ overhang upon hybridization of the second probe to the target RNA.

51. The method of any one of claims 41-50, wherein the capture sequence in the second probe comprises a 3’ polyadenine sequence.

52. The method of any one of claims 41-51, wherein the first probe and the second probe are ligated using the target RNA as a template without gap filling prior to the ligation.

53. The method of any one of claims 41-51, wherein the first probe and the second probe are ligated using the target RNA as a template with gap filling prior to the ligation, wherein the gap filling comprises hybridization of a gap-fill oligonucleotide to the target RNA and / or primer extension of the first probe or the second probe by a polymerase.

54. The method of any one of claims 41-53, wherein the releasing in f) comprises releasing the tethered RNA from the hydrogel matrix.

55. The method of any one of claims 41-54, wherein the releasing in f) comprises releasing the ligation product from the target RNA.

56. The method of any one of claims 41-55, wherein the releasing in f) comprises contacting the hydrogel matrix with an RNase H.

57. The method of any one of claims 41-56, wherein the capture domain in the capture probe comprises a 3’ poly(dT) sequence.

58. The method of any one of claims 41-57, wherein between f) and g), the method comprises generating a spatially labeled polynucleotide comprising (i) from 5’ to 3’, a sequence of the released ligation product and a complementary sequence of the spatial barcode, or (ii) from 5’ to 3’, a sequence of the spatial barcode and a complementary sequence of the released ligation product.

59. The method of claim 58, wherein the spatially labeled polynucleotide or a portion thereof is released from the spatial array for analysis.

60. The method of claim 59, wherein the spatially labeled polynucleotide or portion thereof released from the spatial array is analyzed by nucleic acid sequencing.ny-2785575202412020440 61. The method of any one of claims 41-60, wherein the method comprises imaging the spatial array using fluorescence microscopy.

62. The method of any one of claims 41-61, wherein the method comprises amplifying the released ligation product or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released ligation product or the spatially labeled polynucleotide or an amplification product thereof.

63. The method of any one of claims 41-62, wherein the method comprises performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array.

64. A method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) releasing the target RNA from the hydrogel matrix; d) hybridizing or ligating the released target RNA to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and e) determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the target RNA, or a complement thereof, and a sequence of the spatial barcode, or a complement thereof.

65. The method of claim 64, wherein prior to a), the biological sample is a fresh frozen tissue sample or a decrosslinked tissue sample, or wherein prior to a), the biological sample is a fixed tissue sample, and between a) and b), the biological sample embedded in the hydrogel matrix is decrosslinked.

66. The method of claim 64 or claim 65, wherein the target RNA is 3’ tethered to the hydrogel matrix.

67. The method of claim 64 or claim 65, wherein the target RNA is 5’ tethered to the hydrogel matrix.

68. The method of any one of claims 64-67, wherein the capture domain in the capture probe comprises a 3’ poly(dT) sequence.ny-2785575202412020440 69. The method of any one of claims 64-68, wherein between d) and e), the method comprises generating an extended capture probe comprising, from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released target RNA.

70. The method of claim 69, wherein the extended capture probe further comprises a 3’ end homopolymer sequence.

71. The method of claim 69 or claim 70, further comprising hybridizing a template switch oligonucleotide to the extended capture probe, and extending the extended capture probe using the template switch oligonucleotide as a template to generate a further extended capture probe.

72. The method of claim 71, further comprising hybridizing a primer to the further extended capture probe at a sequence complementary to the template switch oligonucleotide, and extending the primer using the further extended capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a sequence of the released target RNA and a complementary sequence of the spatial barcode.

73. The method of claim 72, wherein the spatially labeled polynucleotide or a portion thereof is released from the further extended capture probe for analysis.

74. The method of claim 73, wherein the spatially labeled polynucleotide or portion thereof released from the spatial array is analyzed by nucleic acid sequencing.

75. The method of any one of claims 64-74, wherein the method comprises imaging the spatial array using fluorescence microscopy.

76. The method of any one of claims 64-75, wherein the method comprises amplifying the released target RNA, the extended capture probe, the further extended capture probe, or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released target RNA, the extended capture probe, the further extended capture probe, or the spatially labeled polynucleotide, or an amplification product thereof.

77. The method of any one of claims 64-76, wherein the method comprises performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array.

78. A method for analyzing a biological sample, the method comprising:ny-2785575202412020440 a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) hybridizing a probe that is substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; d) extending the hybridized probe to form an extended probe, wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe; e) releasing the extended probe from the hydrogel matrix; f) hybridizing or ligating the released extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and g) determining a spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

79. The method of claim 78, wherein prior to a), the biological sample is a fresh frozen tissue sample or a decrosslinked tissue sample, or wherein prior to a), the biological sample is a fixed tissue sample, and between a) and b), the biological sample embedded in the hydrogel matrix is decrosslinked.

80. The method of claim 78 or claim 79, wherein the target RNA is 3’ tethered to the hydrogel matrix.

81. The method of claim 78 or claim 79, wherein the target RNA is 5’ tethered to the hydrogel matrix.

82. The method of any one of claims 78-81, wherein the probe hybridizes to a poly(A) sequence in the target RNA tethered to the hydrogel matrix.

83. The method of any one of claims 78-82, wherein the probe comprises a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence.

84. The method of any one of claims 78-83, wherein the homopolymer sequence is a poly(C) sequence.ny-2785575202412020440 85. The method of any one of claims 78-84, wherein the capture domain in the capture probe comprises a template switch oligonucleotide sequence and a homopolymer sequence complementary to the homopolymer sequence at the 3’ end of the extended probe.

86. The method of claim 85, comprising extending the extended probe using the capture probe as a template, thereby generating a spatially labeled polynucleotide comprising, from 5’ to 3’: a complementary sequence of the released target RNA and a complementary sequence of the spatial barcode.

87. The method of claim 86, wherein the spatially labeled polynucleotide or a portion thereof is released from the capture probe for analysis.

88. The method of claim 87, wherein the spatially labeled polynucleotide or portion thereof released from the spatial array is analyzed by nucleic acid sequencing.

89. The method of any one of claims 78-88, wherein the method comprises imaging the spatial array using fluorescence microscopy.

90. The method of any one of claims 78-89, wherein the method comprises amplifying the released target RNA, the extended probe, the capture probe, or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released target RNA, the extended probe, the capture probe, or the spatially labeled polynucleotide, or an amplification product thereof.

91. The method of any one of claims 78-90, wherein the method comprises performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array.

92. A method for analyzing a biological sample, the method comprising: a) embedding the biological sample in a hydrogel matrix and tethering a target RNA from the biological sample to the hydrogel matrix; b) removing the embedded biological sample from the hydrogel matrix; c) hybridizing a first probe that is substantially complementary to a portion of the target RNA tethered to the hydrogel matrix; d) extending the hybridized probe to form an extended probe, wherein the extension appends a homopolymer sequence at the 3’ end of the extended probe;ny-2785575202412020440 e) hybridizing a second probe to the extended probe in the hydrogel matrix and further extending the extended probe using the second probe as a template to form a further extended probe, wherein the extension comprises incorporating a polyuridine-polyadenine double stranded region at the end of a duplex formed between the second probe and the further extended probe; f) releasing the further extended probe comprising the double stranded region from the hydrogel matrix; g) hybridizing or ligating the released further extended probe to a capture domain of a capture probe on a spatial array, wherein the capture probe further comprises a spatial barcode; and h) determining the spatial transcriptomic profile for the biological sample by determining the sequence of all or a portion of the further extended probe, or a complement thereof, and the sequence of the spatial barcode, or a complement thereof.

93. The method of claim 92, wherein prior to a), the biological sample is a fresh frozen tissue sample or a decrosslinked tissue sample, or wherein prior to a), the biological sample is a fixed tissue sample, and between a) and b), the biological sample embedded in the hydrogel matrix is decrosslinked.

94. The method of claim 92 or claim 93, wherein the target RNA is 3’ tethered to the hydrogel matrix.

95. The method of claim 92 or claim 93, wherein the target RNA is 5’ tethered to the hydrogel matrix.

96. The method of any one of claims 92-95, wherein the first probe hybridizes to a poly(A) sequence in the target RNA tethered to the hydrogel matrix.

97. The method of any one of claims 92-96, wherein the first probe comprises a 3’ poly(dT) sequence and a 5’ non-poly(dT) sequence.

98. The method of any one of claims 92-97, wherein the second probe comprises a 5’ poly(U) sequence, a template switch oligonucleotide sequence, and a homopolymer sequence complementary to the homopolymer sequence at the 3’ end of the extended probe.ny-2785575202412020440 99. The method of any one of claims 92-98, wherein the further extended probe comprises a 3’ poly(A) sequence.

100. The method of any one of claims 92-99, wherein the releasing in f) comprises releasing the tethered RNA from the hydrogel matrix.

101. The method of any one of claims 92-100, wherein the releasing in f) comprises releasing the further extended probe from the target RNA.

102. The method of any one of claims 92-101, wherein the releasing in f) comprises contacting the hydrogel matrix with an RNase H.

103. The method of any one of claims 92-102, wherein the capture domain in the capture probe comprises a 3’ poly(dT) sequence.

104. The method of any one of claims 92-103, wherein between g) and h), the method comprises generating a spatially labeled polynucleotide comprising: i) from 5’ to 3’: a sequence of the spatial barcode and a complementary sequence of the released further extended probe, or ii) from 5’ to 3’: a sequence of the released further extended probe and a complementary sequence of the spatial barcode.

105. The method of claim 104, wherein the spatially labeled polynucleotide or a portion thereof is released from the spatial array for analysis.

106. The method of claim 105, wherein the spatially labeled polynucleotide or portion thereof released from the spatial array is analyzed by nucleic acid sequencing.

107. The method of any one of claims 92-106, wherein the method comprises imaging the spatial array using fluorescence microscopy.

108. The method of any one of claims 92-107, wherein the method comprises amplifying the released further extended probe or the spatially labeled polynucleotide, or detecting on the spatial array an optical signal associated with the released further extended probe or the spatially labeled polynucleotide or an amplification product thereof.

109. The method of any one of claims 92-108, wherein the method comprises performing rolling circle amplification in the embedded biological sample, in the hydrogel matrix with the biological sample removed, or on the spatial array. 162ny-2785575202412020440 110. The method of any one of claims 41-109, wherein the biological sample is contacted with a plurality of transposomes, wherein a transposome of the plurality of transposomes comprises (i) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, (ii) a linker, and (iii) a reactive group, thereby tagmenting an open chromatin in the biological sample with the plurality of transposomes, and wherein the tagmented open chromatin is tethered to the hydrogel matrix via the reactive group of the tagmented open chromatin, wherein after the biological sample is removed from the hydrogel matrix, the tagmented open chromatin is released from the hydrogel matrix, the released tagmented open chromatin is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the tagmented open chromatin or a complement thereof and (ii) the spatial barcode or a complement thereof is determined.

111. The method of any one of claims 41-110, wherein the biological sample is contacted with an antibody that binds to a chromatin protein or chromatin-associated protein in the biological sample, and with a transposome-binding moiety complex that binds to the antibody, wherein the transposome-binding moiety complex comprises (i) a transposase, (ii) an antibody-binding moiety, (iii) a transposon end sequence comprising a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, and (iv) a reactive group, thereby tagmenting a genomic DNA in the biological sample with the transposome-binding moiety complex, wherein the tagmented genomic DNA is tethered to the hydrogel matrix via the reactive group of the transposome-binding moiety complex, wherein after the biological sample is removed from the hydrogel matrix, the tagmented genomic DNA is released from the hydrogel matrix, the released tagmented genomic DNA is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the tagmented genomic DNA or a complement thereof and (ii) the spatial barcode or a complement thereof is determined.ny-2785575202412020440 112. The method of any one of claims 41-111, wherein the biological sample is contacted with an antibody-oligonucleotide conjugate, wherein the antibody-oligonucleotide conjugate comprises (i) an antibody that binds to a target protein in the biological sample, (ii) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on the spatial array, (iii) a barcode sequence associated with the antibody, and (iv) a reactive group, wherein the antibody-oligonucleotide conjugate is tethered to the hydrogel matrix via the reactive group of the antibody-oligonucleotide conjugate, wherein after the biological sample is removed from the hydrogel matrix, the antibody- oligonucleotide conjugate or a portion thereof is released from the hydrogel matrix, the capture sequence of the released antibody-oligonucleotide conjugate or portion thereof is hybridized or ligated to a capture domain of a capture probe on the spatial array, and the capture probe further comprises a spatial barcode, and wherein the sequence of (i) the barcode sequence associated with the antibody or a complement thereof and (ii) the spatial barcode or a complement thereof is determined.

113. The method of any one of claims 110-112, wherein two or more or all of the released ligation product in claim 41, the released target RNA in claim 64, the released extended probe in claim 78, the released further extended probe in claim 91, the released tagmented open chromatin in claim 109, the released tagmented genomic DNA in claim 110, the released antibody- oligonucleotide conjugate or portion thereof in claim 111 are hybridized or ligated to capture probes comprising capture domains of different sequences on the spatial array.

114. A method for analyzing a biological sample, comprising: a) contacting the biological sample with a plurality of transposomes, wherein a transposome of the plurality comprises (i) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, (ii) a linker, and (iii) a reactive group; b) tagmenting an open chromatin in the biological sample with the plurality of transposomes; c) embedding the biological sample in a hydrogel matrix, wherein the tagmented open chromatin is tethered to the hydrogel matrix via the reactive group of the tagmented open chromatin;ny-2785575202412020440 d) removing the biological sample from the hydrogel matrix; e) releasing the tagmented open chromatin from the hydrogel matrix, wherein the released tagmented open chromatin is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and f) determining a profile of the open chromatin in the biological sample by determining the sequence of (i) the tagmented open chromatin or a complement thereof and (ii) the spatial barcode or a complement thereof.

115. Th method of claim 114, wherein the linker comprises an antibody-binding moiety, optionally wherein the antibody-binding moiety is protein A, protein G, or a fusion protein thereof; and / or wherein the capture sequence is in a first transposon end sequence, and the transposome or the transposome-binding moiety complex comprise a second transposon end sequence comprising a functional sequence, optionally wherein the functional sequence comprises a primer binding sequence or a complement thereof.

116. A method for analyzing a biological sample, comprising: a) contacting the biological sample with: an antibody that binds to a chromatin protein or chromatin-associated protein in the biological sample, a transposome-binding moiety complex that binds to the antibody, wherein the transposome-binding moiety complex comprises (i) a transposase, (ii) an antibody-binding moiety, (iii) a transposon end sequence comprising a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, and (iv) a reactive group; b) tagmenting a genomic DNA in the biological sample with the transposome- binding moiety complex; c) embedding the biological sample in a hydrogel matrix, wherein the tagmented genomic DNA is tethered to the hydrogel matrix via the reactive group of the transposome- binding moiety complex; d) removing the biological sample from the hydrogel matrix;ny-2785575202412020440 e) releasing the tagmented genomic DNA from the hydrogel matrix, wherein the released tagmented genomic DNA is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and f) determining the sequence of (i) the tagmented genomic DNA or a complement thereof and (ii) the spatial barcode or a complement thereof.

117. The method of claim 116, wherein the chromatin protein or chromatin-associated protein is a histone or a non-histone DNA binding protein, optionally wherein the non-histone DNA binding protein comprises a transcription factor or a transcription co-factor.

118. The method of claim 116 or claim 117, wherein the antibody binds to one or more epigenetic modifications, optionally wherein the antibody binds to one or more histone modifications.

119. The method of any one of claims 116-118, wherein the antibody directly binds to the chromatin protein or chromatin-associated protein, and the antibody-binding moiety in the transposome-binding moiety complex directly binds to the antibody.

120. The method of any one of claims 116-118, wherein the antibody is a primary antibody that directly binds to the chromatin protein or chromatin-associated protein, the biological sample is contacted with a secondary antibody that directly binds to the primary antibody, and the antibody-binding moiety in the transposome-binding moiety complex directly binds to the secondary antibody.

121. A method for analyzing a biological sample, comprising: a) contacting the biological sample with an antibody-oligonucleotide conjugate comprising (i) an antibody that binds to a target protein in the biological sample, (ii) a capture sequence configured to be hybridized or ligated to a capture domain of a capture probe on a spatial array, (iii) a barcode sequence associated with the antibody, and (iv) a reactive group; b) embedding the biological sample in a hydrogel matrix, wherein the antibody- oligonucleotide conjugate is tethered to the hydrogel matrix via the reactive group of the antibody-oligonucleotide conjugate; c) removing the biological sample from the hydrogel matrix; d) releasing the antibody-oligonucleotide conjugate or a portion thereof from the hydrogel matrix, wherein capture sequence of the released antibody-oligonucleotide conjugate or 166ny-2785575202412020440 portion thereof is hybridized or ligated to a capture domain of a capture probe on the spatial array, and wherein the capture probe further comprises a spatial barcode; and e) determining the sequence of (i) the barcode sequence associated with the antibody or a complement thereof and (ii) the spatial barcode or a complement thereof.

122. The method of any one of claims 110-121, wherein the reactive group comprises a 5’ acrylic phosphoramidite.

123. The method of any one of claims 110-122, wherein the transposome, the transposome- binding moiety complex, and / or the antibody-oligonucleotide conjugate comprises a cleavable linker connected to the reactive group.

124. The method of any one of claims 110-123, wherein the transposome, the transposome- binding moiety complex, and / or the antibody-oligonucleotide conjugate comprises a functional sequence.

125. The method of claim 124, wherein the functional sequence comprises a primer binding sequence or a complement thereof.ny-2785575

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