RNA probing and amplification methods for single cell analysis on fixed cells

Abstract

Disclosed herein include systems, methods, compositions, and kits for determining the spatial location and / or copy number of a nucleic acid target in a fixed sample. There are provided, in some embodiments, probing oligonucleotides comprising a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and / or a predetermined spatial label. The method can comprise contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with said probing oligonucleotides. The method can comprise extending in situ the probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides. The method can comprise barcoding the plurality of extended probing oligonucleotides using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides.

Description

RNA PROBING AND AMPLIFICATION METHODS FOR SINGLE CELL ANALYSISON FIXED CELLSRELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Ser. No. 63 / 503,856, filed May 23, 2023, the content of this related application is incorporated herein by reference in its entirety for all purposes.BACKGROUNDField

[0002] The present disclosure relates generally to the field of molecular biology, for example determining gene expression using molecular barcoding.Description of the Related Art

[0003] Many cell samples comprise fixed cells, including patient samples that are required to be stored in fixatives. However, current gene expression profiling compositions and methods, including single cell RNA sequencing workflows and platforms, are not compatible with fixed samples because mRNAs are cross-linked and not available for capture by conventional mRNA capture methods (e.g., via poly A / dT capture). In situ hybridization and the FISH method have been visualize mRNAs in fixed samples; however, the number of probes that can be used to detect mRNAs is limited due to the limited number of fluorescent detectors, rendering transcriptome-level analysis difficult. There is a need for compositions, methods, systems, and kits capable of analyzing gene expression in fixed samples. Moreover, there is a need for compositions, methods, systems, and kits for spatial analysis of gene expression in fixed samples.SUMMARY

[0004] Disclosed herein include methods for labeling nucleic acid targets in a sample. In some embodiments, the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality7of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary7to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality7of barcoded probing oligonucleotides, wherein each oligonucleotide barcodeof the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target. The method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.

[0005] Disclosed herein include methods for determining the copy number of a nucleic acid target in a sample. In some embodiments, the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality' of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target. The method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.

[0006] Disclosed herein include methods for determining the spatial location and copy number of a nucleic acid target in a sample. In some embodiments, the method comprises: contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and a predetermined spatial label. In some embodiments probing oligonucleotides contacted with the same spatial location comprise the same spatial label sequence, and wherein probingoligonucleotides contacted with distinct spatial locations of the sample comprise different spatial label sequences. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality' of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality' of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target. The method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a nucleic acid target to determine the copy number of the nucleic acid target at each spatial location of the sample.

[0007] In some embodiments, barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality' of oligonucleotide barcodes comprises: providing a coupling oligonucleotide comprising a 5 ’ complement of the coupling sequence and a 3’ complement of a capture sequence; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5’ complement of the coupling sequence of the coupling oligonucleotide; hybridizing the 3’ complement of the capture sequence of the coupling oligonucleotide with a capture sequence of an oligonucleotide barcode of the plurality of oligonucleotide barcodes; and / or ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode. The method can comprise: before ligating the extended probing oligonucleotide to the oligonucleotide barcode, filling a gap between the extended probing oligonucleotide and the hybridized oligonucleotide barcode with a DNA polymerase lacking at least one of 5’ to 3’ exonuclease activity and 3’ to 5’ exonuclease activity. In some embodiments, ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode is performed with a DNA ligase. In some embodiments, the coupling oligonucleotide is a single-stranded oligonucleotide. In some embodiments, the coupling oligonucleotide comprises at least 6 nucleotides. In some embodiments, the coupling sequence comprises at least 4 nucleotides. In some embodiments, the 5’ end of each probing oligonucleotide is phosphorylated. In some embodiments, the probing oligonucleotides are capable of entering acell and / or a nucleus of the sample (e.g., a permeabilized cell and / or a permeabilized nucleus of the sample). The method can comprise: after contacting the probing oligonucleotides with the sample, removing one or more probing oligonucleotides of the plurality of probing oligonucleotides that are not contacted with the sample, optionally removing the one or more probing oligonucleotides not contacted with the sample comprises: removing the one or more probing oligonucleotides that have not entered a cell of the sample. In some embodiments, the contacting step comprises contacting the sample with a device configured to deposit probing oligonucleotides (e.g., an ink jet device). In some embodiments, the device is a needle, a needle array, a tube, a suction device, an injection device, an electroporation device, a fluorescent activated cell sorter device, an ink jet device, a microfluidic device, or any combination thereof. In some embodiments, the device contacts distinct spatial locations of the sample at a specified rate. In some embodiments, the spatial label is 6-60 nucleotides in length. In some embodiments, said two or more spatial locations comprise at least about 3, about 4, about 5. about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, distinct spatial locations of the sample. In some embodiments, a spatial location of the sample corresponds to an area comprising no more than about 50 cells, about 45 cells, about 40 cells, about 35 cells, about 30 cells, about 25 cells, about 20 cells, about 15 cells, about 10 cells, about 9 cells, about 8 cells, about 7 cells, about 6 cells, about 5 cells, about 4 cells, about 3 cells, about 2 cells, or about 1 cell.

[0008] The method can comprise: contacting the sample with extension reagents. In some embodiments, at least a portion of the contacting step is performed in the presence in the presence of extension reagents. In some embodiments, the entire contacting step is performed in the presence in the presence of the extension reagents. In some embodiments, the contacting and extension steps are simultaneous. In some embodiments, the extension is performed in situ. In some embodiments, said extension comprises in situ reverse transcription. In some embodiments, cells of the sample remain intact during the extension step. In some embodiments, the extension reagents comprise reverse transcription reagents. In some embodiments, reverse transcription reagents comprise a reverse transcriptase and dNTPs. In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase. In some embodiments, the viral reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase or a Moloney murine leukemia virus (MMLV) reverse transcriptase.

[0009] In some embodiments, the sample is physically divided or is intact during the contacting step. In some embodiments, the sample comprises a single cell. In some embodiments, the sample comprises a plurality of single cells. In some embodiments, the samplecomprises a plurality of cells, and optionally the method comprises: disassociating the sample to generate a plurality of single cells, optionally said disassociating comprises chemical dissociating, enzy matic dissociating, and / or mechanical dissociating, optionally said disassociating employs one or more of collagenase, chymotrypsin, dispase, elastase, hyaluronidase, pancreatin, papain, and trypsin. The method can comprise: prior to the barcoding step: partitioning the plurality of single cells to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of single cells; and in the partition comprising the single cell, contacting the extended probing oligonucleotide with the plurality of oligonucleotide barcodes. In some embodiments, in the partition comprising the single cell, contacting the single cell with a lysis buffer at 15-65 °C to lyse the single cell. In some embodiments, the lysis buffer comprises an agent capable of dissociating protein-nucleic acid complexes.

[0010] In some embodiments, each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a first universal sequence. In some embodiments, obtaining sequencing data comprises: amplifying the plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or complement thereof, and amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, thereby generating a plurality of amplified barcoded probing oligonucleotides, wherein obtaining sequencing data comprises obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides, or products thereof. In some embodiments, obtaining sequencing data comprises attaching the binding sites of sequencing primers and / or sequencing adaptors to the plurality of barcoded probing oligonucleotides, or products thereof. In some embodiments, the amplification primer(s) comprise a second universal sequence and / or wherein the first primer comprises a third universal sequence. In some embodiments, the first universal sequence, the second universal sequence, and / or the third universal sequence are the same. In some embodiments, the first universal sequence, the second universal sequence, and / or the third universal sequence are different. In some embodiments, the first universal sequence, the second universal sequence, and / or the third universal sequence comprise the binding sites of sequencing primers and / or sequencing adaptors, complementary' sequences thereof, and / or portions thereof. In some embodiments, the sequencing adaptors comprise a P5 sequence, a P7 sequence, complementary sequences thereof, and / or portions thereof. In some embodiments, the sequencing primers comprise a Read 1 sequencing primer, a Read 2 sequencing primer, complementary sequences thereof, and / or portions thereof.

[0011] In some embodiments, the sample comprises a plurality of nucleic acidtargets, such as, for example, a target panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350. about 375, about 400, about 425, about 450, about 475, or about 500, distinct nucleic acid targets. In some embodiments, two or more nucleic acid targets of the target panel are biomarkers. In some embodiments, the biomarkers are biomarkers for a disease or condition. In some embodiments, the disease or condition is a cancer, an infection, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a bacterial infection, or any combination thereof. In some embodiments, the contacting step comprises contacting the sample with a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality comprises a probe sequence configured hybridize a nucleic acid target of the plurality of nucleic acid targets. In some embodiments, determining the copy number of the nucleic acid target in the sample comprises determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of molecular labels with distinct sequences associated with the plurality of barcoded probing oligonucleotides, or products thereof, comprising a sequence of the each of the plurality of nucleic acid targets. The method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with each of the plurality of nucleic acid targets to determine the copy number of each of the plurality of nucleic acid targets at each spatial location of the sample. In some embodiments, the amplification primer(s) comprise a panel of amplification primers configured to hybridize the plurality of nucleic acid targets, or complements thereof, such as, for example, a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80. about 90, about 100. about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500, distinct amplification primers. In some embodiments, the nucleic acid target comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation product, RNA comprising a poly(A) tail, a sample indexing oligonucleotide, a cellular component-binding reagent specific oligonucleotide, or any combination thereof.

[0012] In some embodiments, the plurality of cells comprise one or more cell types. In some embodiments, said one or more cell types are selected from the group consisting of:brain cells, heart cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, and circulatory cells, or any combination thereof. In some embodiments, the sample comprises a biological sample, a clinical sample, an environmental sample, a biological fluid, a tissue, a tissue section derived from a subject, or any combination thereof. In some embodiments, the subject is a human, a mouse, a dog, a rat. or a vertebrate. The method can comprise: determining genotype, phenotype, or one or more genetic mutations of the subject based on the spatial location of the nucleic acid targets in the sample. The method can comprise: predicting susceptibility of the subject to one or more diseases, such as, for example, a cancer or a hereditary disease. The method can comprise: determining cell types of the plurality of cells in the sample. In some embodiments, a drug is chosen based on predicted responsiveness of the cell types of the plurality of cells in the sample. The method can comprise: imaging the sample, optionally imaging the sample before the contacting step and / or after the contacting step, optionally the imaging generates imaging data. In some embodiments, imaging the sample comprises staining the sample with a stain, wherein the stain is a fluorescent stain, a negative stain, an antibody stain, or any combination thereof. In some embodiments, staining comprises Immunocytochemistry (ICC), Immunohistochemistry (IHC), Immunofluorescence (IF), or any combination thereof. In some embodiments, imaging comprises microscopy, confocal microscopy, time-lapse imaging microscopy, fluorescence microscopy, multi-photon microscopy, quantitative phase microscopy, surface enhanced Raman spectroscopy, videography, manual visual analysis, automated visual analysis, or any combination thereof. The method can comprise: associating the imaging data and sequencing data of one or more spatial locations of the sample. The method can comprise: correlation analysis of the imaging data and the sequencing data of the spatial locations. In some embodiments, the correlation analysis identifies one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of therapeutic agents, and / or cellular targets of candidate therapeutic agents. In some embodiments, said imaging produces an image that is used to construct a map of a physical representation of said sample. In some embodiments, said map is two dimensional or three dimensional. The method can comprise: mapping the nucleic acid targets and / or cellular component targets onto the map of the sample. The method can comprise: mapping one or more single cells of the plurality of cells onto the map of the sample.

[0013] In some embodiments, the sample has been contacted with one or more fixing agents and / or permeabilizing agents. In some embodiments, the sample comprises a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof. In some embodiments, the sample comprises a fresh tissue section, a frozen tissue section, a fixed tissue section, aformalin-fixed tissue section, a formalin-fixed paraffin-embedded (FFPE) tissue section, an acetone fixed tissue section, a paraformaldehyde (PF A) fixed tissue section, and / or a methanol fixed tissue section. In some embodiments, the sample comprises a nuclei suspension, such as, for example, a fixed nuclei suspension and / or a permeabilized nuclei suspension. In some embodiments, the sample comprises cell(s), such as, for example, fresh cell(s), frozen cell(s), fixed cell(s), formalin-fixed cell(s), formalin-fixed paraffin-embedded (FFPE) cell(s), acetone fixed cell(s), a paraformaldehyde (PF A) fixed cell(s), and / or a methanol fixed cell(s). The method can comprise: permeabilizing the sample and / or fixing the sample. In some embodiments, fixing the sample comprises contacting the sample with a fixing agent. In some embodiments, the fixing agent comprises a non-cross-linking fixative (e.g., methanol). In some embodiments, the fixing agent comprises a cross-linking agent. In some embodiments, the crosslinking agent comprises a cleavable cross-linking agent. In some embodiments, the cleavable cross-linking agent comprises or is derived from dithiobis(succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), Bis |2-(Succinimidooxycarbonyloxy)ethyl] Sulfone (BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), dimethyl 3,3'-dithiobispropionimidate (DTBP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2- pyridyldithio)propionamido)hexanoate (LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl- a(2-pyridyldithio)toluene (SMPT). 3-(2-pyridyldithio)propionyl hydrazide (PDPH), succinimidyl 2-((4,4'-azipentanamido)ethyl)-l,3'-dithiopropionate (SDAD, NHS-SS-Diazirine), or any combination thereof. In some embodiments, the cleavable cross-linking agent comprises a cleavable linkage selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, an acid labile linker, a heat sensitive linkage, an enzymatically cleavable linkage, and a combination thereof. In some embodiments, the cleavable cross-linking agent is a thiol-cleavable cross-linking agent or comprises a disulfide linker. In some embodiments, the fixing agent comprises paraformaldehyde (PFA), dithiobis(succinimidyl propionate (DSP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), CellCover, or a combination thereof. In some embodiments, fixing the sample and permeabilizing the sample are carried out simultaneously. In some embodiments, fixing and permeabilizing the sample is carried out in the presence of a dual function agent capable of fixing and permeabilizing the sample. In some embodiments, the dual functional agent is methanol.

[0014] In some embodiments, permeabilizing the sample comprises contacting the sample with a permeabilizing agent. The method can comprise: after contacting the plurality of probing oligonucleotides or the plurality of cellular component-binding reagents with the sample, removing the permeabilizing agent from the sample. In some embodiments, the permeabilizing agent is capable of (i) permeabilizing the cell membrane of the cell(s), (ii)making a cell membrane of the cell(s) permeable to the probing oligonucleotides or the cellular component-binding reagents, or both. In some embodiments, the permeabilizing agent comprises (i) a solvent, a detergent, or a surfactant; (ii) BD Cytoperm; (iii) a saponin or a derivative thereof; (iv) Triton X-100, (v) methanol or a derivative thereof, and / or (vi) digitonin or a derivative thereof. In some embodiments, the agent capable of dissociating protein-nucleic acid complexes comprises a broad-spectrum serine protease. In some embodiments, the broadspectrum serine protease is proteinase K. In some embodiments, the lysis buffer comprises an unfixing agent. In some embodiments, the unfixing agent comprises a thiol, hydoxylamine, periodate, a base, or any combination thereof. In some embodiments, the lysis buffer comprises DTT. The method can comprise: reversing the fixation of the sample and / or single cells. In some embodiments, reversing the fixation of the sample and / or single cells comprises UV photocleaving, chemical treatment, heating, enzy me treatment, or any combination thereof.

[0015] The sample can comprise a plurality of cellular component targets, and the method further comprises: contacting a plurality of cellular component-binding reagents with the sample, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular componentbinding reagent is capable of specifically binding to at least one of the plurality of cellular component targets; barcoding the cellular component-binding reagent specific oligonucleotides to generate a plurality of barcoded cellular component-binding reagent specific oligonucleotides each comprising a sequence complementary' to at least a portion of the unique identifier sequence and a molecular label sequence: and obtaining sequencing data comprising a plurality’ of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and at least a portion of the unique identifier sequence. In some embodiments, obtaining sequencing data comprises attaching the binding sites of sequencing primers and / or sequencing adaptors to the barcoded cellular component-binding reagent specific oligonucleotides, or products thereof.

[0016] The method can comprise: after contacting the plurality7of cellular component-binding reagents with the sample, removing one or more cellular component-binding reagents of the plurality of cellular component-binding reagents that are not contacted with the sample, optionally removing the one or more cellular component-binding reagents not contacted with the sample comprises: removing the one or more cellular component-binding reagents not contacted with the respective at least one of the plurality of cellular component targets. In some embodiments, the cellular component target comprises an intracellular protein, a carbohydrate, alipid, a protein, an extracellular protein, a cell-surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof. In some embodiments, the cellular component-binding reagent specific oligonucleotide comprises a second molecular label, optionally at least ten of the plurality of cellular component-binding reagent specific oligonucleotides comprise different second molecular label sequences. In some embodiments, the second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides are different, and wherein the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides are identical. In some embodiments, the second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides are different, and wherein the unique identifier sequences of the at least two cellular componentbinding reagent specific oligonucleotides are different. In some embodiments, the number of unique molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample. In some embodiments, the number of unique second molecular label sequences associated with the unique identifier sequence for the cellular componentbinding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample.

[0017] The method can comprise: prior to contacting a plurality of cellular component-binding reagents with the sample and / or contacting the plurality of probing oligonucleotides with the sample, contacting the sample with a blocking reagent, one or more decoy oligonucleotides, and / or one or more blocking oligonucleotides. In some embodiments, contacting a plurality of cellular component-binding reagents with the sample is conducted in the presence of a blocking reagent. In some embodiments, the blocking reagent comprises a plurality of oligonucleotides complementary to at least a portion of the cellular componentbinding reagent specific oligonucleotides. In some embodiments, the blocking reagent comprises an antibody or a fragment thereof derived from a first species, and wherein the blocking reagent comprises sera derived from the first species. In some embodiments, the sample comprises one or more non-target nucleic acids, wherein the blocking reagent comprises a plurality’ of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acids. In some embodiments, each of the plurality of decoy oligonucleotides are capable of hybridizing to at least a portion of a non-target nucleic acid. In some embodiments, the decoy oligonucleotide: comprises a sequence complementary to at least a portion of a non-targetnucleic acid; comprises a sequence identical to or substantially similar to a sequence of the cellular component-binding reagents specific oligonucleotides, optionally the sequence is 3-40 nucleotides in length; has at most 50% sequence identity to the cellular component-binding reagent specific oligonucleotides; does not comprise a UMI; comprises a random sequence, and optionally the random sequence is about four, five, six, seven, eight, nine, ten. eleven, twelve, thirteen, fourteen, or fifteen nucleotides in length; does not comprise any sequence having more than four, five, six, or seven consecutive Ts or As; comprise at least one G or C in every four, five, six. or seven consecutive nucleotides; comprises one or more modified nucleotides; comprises a 5’ modification, and optionally the 5?modification comprises a 5' Amino Modifier C12 modification (5AmMC12); comprises a 3’ modification, and optionally the 3’ modification comprises a 3' dideoxy-C modification (ddC); and / or is 30 to 65 nucleotides in length. In some embodiments, the sample comprises one or more undesirable nucleic acid species, the method comprising: contacting a blocking oligonucleotide with the sample, wherein the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleic acid species; whereby the reverse transcription of the at least one of the one or more undesirable nucleic acid species is reduced by the blocking oligonucleotide. In some embodiments, the blocking oligonucleotide: is contacted with the sample before the plurality of probing oligonucleotides is contacted with the sample; is contacted with the sample after the plurality of probing oligonucleotides is contacted with the sample; and / or is contacted with the sample when the plurality of probing oligonucleotides is contacted with the sample. The method can comprise: providing blocking oligonucleotides that specifically bind to two or more undesirable nucleic acid species, optionally at least 10 or to at least 100 undesirable nucleic acid species, in the sample. In some embodiments, the blocking oligonucleotide: is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera; specifically binds to within 100 nt, to within 50 nt, or to within 25 nt of the 3’ end of the one or more undesirable nucleic acid species; specifically binds to within 100 nt of the 5‘ end of the one or more undesirable nucleic acid species, or the blocking oligonucleotide specifically binds to within 100 nt of the middle of the one or more undesirable nucleic acid species; comprises or does not comprise non-natural nucleotides; has a Tm of at least 50 °C, of at least 60 °C, or of at least 70 °C; is unable to function as a primer for a reverse transcriptase or a polymerase; and / or is 8 nt to 100 nt long, is 10 nt to 50 nt long, is 12 nt to 21 nt long, is 20 nt to 30 nt long, or is about 25 nt long. In some embodiments, the one or more undesirable nucleic acid species amounts to about 50%, to about 60%, to about 70%, or to about 80% of the nucleic acid content of the sample. In some embodiments, the undesirable nucleic acid species is selected from the group consisting of ribosomal RNA, mitochondrial RNA, genomic DNA,intronic sequence, high abundance sequence, and a combination thereof. In some embodiments, the one or more undesirable nucleic acid species are mRNA molecules and the blocking oligonucleotide specific binds to within 10 nt of the 3’ poly (A) tail of the one or more undesirable nucleic acid species.

[0018] In some embodiments, each molecular label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides. In some embodiments, each capture sequence of the plurality of oligonucleotide barcodes comprises at least 4 nucleotides. In some embodiments, the plurality of oligonucleotide barcodes are associated with a solid support, and wherein a partition of the plurality of partitions comprises a single solid support. In some embodiments, the plurality of oligonucleotide barcodes each comprise a cell label. In some embodiments, each cell label of the plurality' of oligonucleotide barcodes comprises at least 6 nucleotides. In some embodiments, oligonucleotide barcodes of the plurality' of oligonucleotide barcodes associated with the same solid support comprise the same cell label. In some embodiments, oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with different solid supports comprise different cell labels. In some embodiments, the solid support comprises a synthetic particle, a planar surface, or a combination thereof. The method can comprise: associating a synthetic particle comprising the plurality of oligonucleotide barcodes with the cell in the partition. The method can comprise: lysing the cell after associating the synthetic particle with the cell. In some embodiments, lysing the cell comprises heating the cell, contacting the cell with a detergent, changing the pH of the cell, or any combination thereof. In some embodiments, the synthetic particle and the single cell are in the same partition, and optionally the partition is a well or a droplet. In some embodiments, at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is immobilized or partially immobilized on the synthetic particle, or at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is enclosed or partially enclosed in the synthetic particle. In some embodiments, the synthetic particle is disruptable (e.g., a disruptable hydrogel particle). In some embodiments, the synthetic particle comprises a bead. In some embodiments, the bead comprises a Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A / G conjugated bead, a protein L conjugated bead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or any combination thereof. In some embodiments, the synthetic particle comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. In some embodiments, eacholigonucleotide barcode of the plurality of oligonucleotide barcodes comprises a linker functional group. In some embodiments, the synthetic particle comprises a solid support functional group. In some embodiments, the support functional group and the linker functional group are associated with each other, and optionally the linker functional group and the support functional group are individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), and any combination thereof.

[0019] Disclosed herein include compositions (e.g., kits). In some embodiments, the kit comprises: a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize a nucleic acid target, optionally the probing oligonucleotides comprise a predetermined spatial label; a coupling oligonucleotide comprising a 5’ complement of the coupling sequence and a 3’ complement of a capture sequence; a plurality of oligonucleotide barcodes, wherein the 3‘ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes is associated with a solid support, wherein the 5’ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a capture sequence; a first primer capable of hybridizing to the first universal sequence, optionally further comprising a third universal sequence; amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, optionally further comprising a second universal sequence; a DNA ligase; extension reagents, optionally reverse transcription reagents, further optionally a reverse transcriptase and dNTPs; one or more fixing agents; one or more permeabilizing agents; a cross-linking agent; unfixing agent: lysis buffer; a plurality of cellular component-binding reagents, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is capable of specifically binding to at least one of a plurality of cellular component targets; a blocking reagent; one or more decoy oligonucleotides; and / or one or more blocking oligonucleotides.

[0020] In some embodiments, the plurality of probing oligonucleotides comprises a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality comprises a probe sequence configured hybridize a nucleic acid target of a plurality of nucleic acid targets, optionally a target panel of at least about 2, about 3. about 4, about 5, about 6. about 7, about 8, about 9, about 10, about 12. about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, or about 500. distinct nucleic acid targets. In some embodiments, the amplification primer(s)comprise a panel of amplification primers configured to hybridize a plurality of nucleic acid targets, or complements thereof, optionally a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90. about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325. about 350, about 375, about 400, about 425, about 450, about 475, or about 500, distinct amplification primers.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 illustrates a non-limiting exemplary barcode.

[0022] FIG. 2 shows a non-limiting exemplary’ workflow of barcoding and digital counting.

[0023] FIG. 3 is a schematic illustration showing a non-limiting exemplary process for generating an indexed library of targets barcoded at the 3’-ends from a plurality' of targets.

[0024] FIGS. 4A-4D depict a non-limiting exemplary' schematic workflow for gene expression analysis of fixed cells.DETAILED DESCRIPTION

[0025] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.

[0026] All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety' with respect to the related technology’.

[0027] Quantifying small numbers of nucleic acids, for example messenger ribonucleotide acid (mRNA) molecules, is clinically important for determining, for example, the genes that are expressed in a cell at different stages of development or under different environmental conditions. However, it can also be very’ challenging to determine the absolute number of nucleic acid molecules (e.g., mRNA molecules), especially when the number of molecules is very’ small. One method to determine the absolute number of molecules in a sampleis digital polymerase chain reaction (PCR). Ideally, PCR produces an identical copy of a molecule at each cycle. However, PCR can have disadvantages such that each molecule replicates with a stochastic probability, and this probability varies by PCR cycle and gene sequence, resulting in amplification bias and inaccurate gene expression measurements. Stochastic barcodes with unique molecular labels (also referred to as molecular indexes (Mis)) can be used to count the number of molecules and correct for amplification bias. Stochastic barcoding, such as the Precise™ assay (Cellular Research, Inc. (Palo Alto, CA)) and Rhapsody™ assay (Becton, Dickinson and Company (Franklin Lakes, NJ)), can correct for bias induced by PCR and library preparation steps by using molecular labels (MLs) to label mRNAs during reverse transcription (RT).

[0028] The Precise™ assay can utilize a non-depleting pool of stochastic barcodes with large number, for example 6561 to 65536, unique molecular label sequences on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs in a sample during the RT step. A stochastic barcode can comprise a universal PCR priming site. During RT, target gene molecules react randomly with stochastic barcodes. Each target molecule can hybridize to a stochastic barcode resulting to generate stochastically barcoded complementary' ribonucleotide acid (cDNA) molecules). After labeling, stochastically barcoded cDNA molecules from microwells of a microwell plate can be pooled into a single tube for PCR amplification and sequencing. Raw sequencing data can be analyzed to produce the number of reads, the number of stochastic barcodes with unique molecular label sequences, and the numbers of mRNA molecules.

[0029] Disclosed herein include methods for labeling nucleic acid targets in a sample. In some embodiments, the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality' of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing readscomprises a molecular label sequence and a subsequence of the nucleic acid target. The method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.

[0030] Disclosed herein include methods for determining the copy number of a nucleic acid target in a sample. In some embodiments, the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality7of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality’ of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target. The method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality’ of barcoded probing oligonucleotides, or products thereof.

[0031] Disclosed herein include methods for determining the spatial location and copy number of a nucleic acid target in a sample. In some embodiments, the method comprises: contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and a predetermined spatial label. In some embodiments probing oligonucleotides contacted with the same spatial location comprise the same spatial label sequence, and wherein probing oligonucleotides contacted with distinct spatial locations of the sample comprise different spatial label sequences. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality’ of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probingoligonucleotides. or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality7of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality7of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality7of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target. The method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a nucleic acid target to determine the copy number of the nucleic acid target at each spatial location of the sample.Definitions

[0032] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by7one of ordinary skill in the art to w hich the present disclosure belongs. See, e.g., Singleton et al., Dictionary7of Microbiology7and Molecular Biology 2nd ed., J. Wiley & Sons (New7York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory7Manual. Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below7.

[0033] As used herein, the term “adaptor” can mean a sequence to facilitate amplification or sequencing of associated nucleic acids. The associated nucleic acids can comprise target nucleic acids. The associated nucleic acids can comprise one or more of spatial labels, target labels, sample labels, indexing label, or barcode sequences (e.g., molecular labels). The adaptors can be linear. The adaptors can be pre-adenylated adaptors. The adaptors can be double- or single-stranded. One or more adaptor can be located on the 5’ or 3’ end of a nucleic acid. When the adaptors comprise known sequences on the 5’ and 3‘ ends, the known sequences can be the same or different sequences. An adaptor located on the 5’ and / or 3’ ends of a polynucleotide can be capable of hybridizing to one or more oligonucleotides immobilized on a surface. An adaptor can, in some embodiments, comprise a universal sequence. A universal sequence can be a region of nucleotide sequence that is common to tw o or more nucleic acid molecules. The two or more nucleic acid molecules can also have regions of different sequence. Thus, for example, the 5’ adaptors can comprise identical and / or universal nucleic acid sequences and the 3’ adaptors can comprise identical and / or universal sequences. A universal sequence that may be present in different members of a plurality7of nucleic acid molecules can allow the replication or amplification of multiple different sequences using a single universalprimer that is complementary to the universal sequence. Similarly, at least one, two (e.g., a pair) or more universal sequences that may be present in different members of a collection of nucleic acid molecules can allow the replication or amplification of multiple different sequences using at least one, two (e.g.. a pair) or more single universal primers that are complementary to the universal sequences. Thus, a universal primer includes a sequence that can hybridize to such a universal sequence. The target nucleic acid sequence-bearing molecules may be modified to attach universal adaptors (e.g., non-target nucleic acid sequences) to one or both ends of the different target nucleic acid sequences. The one or more universal primers attached to the target nucleic acid can provide sites for hybridization of universal primers. The one or more universal primers attached to the target nucleic acid can be the same or different from each other.

[0034] As used herein the term “associated” or “associated with” can mean that two or more species are identifiable as being co-located at a point in time. An association can mean that two or more species are or were within a similar container. An association can be an informatics association. For example, digital information regarding two or more species can be stored and can be used to determine that one or more of the species were co-located at a point in time. An association can also be a physical association. In some embodiments, two or more associated species are “tethered”, “attached”, or “immobilized” to one another or to a common solid or semisolid surface. An association may refer to covalent or non-covalent means for attaching labels to solid or semi-solid supports such as beads. An association may be a covalent bond between a target and a label. An association can comprise hybridization between two molecules (such as a target molecule and a label).

[0035] As used herein, the term “complementary” can refer to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial.” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. A first nucleotide sequence can be said to be the “complement” of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence can be said to be the “reverse complement” of a second sequence, if the first nucleotide sequence is complementary to a sequence that is the reverse (i.e., the order of the nucleotides is reversed) of the second sequence. As used herein, a “complementary” sequence can refer to a “complement” or a “reverse complement” of a sequence. It is understood from the disclosure that if a molecule can hybridize to another molecule it may be complementary, or partially complementary’, to themolecule that is hybridizing.

[0036] As used herein, the term ’‘digital counting” can refer to a method for estimating a number of target molecules in a sample. Digital counting can include the step of determining a number of unique labels that have been associated with targets in a sample. This methodology, which can be stochastic in nature, transforms the problem of counting molecules from one of locating and identifying identical molecules to a series of yes / no digital questions regarding detection of a set of predefined labels.

[0037] As used herein, the term “label” or “labels” can refer to nucleic acid codes associated with a target within a sample. A label can be, for example, a nucleic acid label. A label can be an entirely or partially amplifiable label. A label can be entirely or partially sequencable label. A label can be a portion of a native nucleic acid that is identifiable as distinct. A label can be a known sequence. A label can comprise a junction of nucleic acid sequences, for example a junction of a native and non-native sequence. As used herein, the term “label” can be used interchangeably with the terms, “index”, “tag,” or “label-tag.” Labels can convey information. For example, in various embodiments, labels can be used to determine an identity of a sample, a source of a sample, an identity of a cell, and / or a target.

[0038] As used herein, the term “non-depleting reservoirs” can refer to a pool of barcodes (e.g., stochastic barcodes) made up of many different labels. A non-depleting reservoir can comprise large numbers of different barcodes such that when the non-depleting reservoir is associated with a pool of targets each target is likely to be associated with a unique barcode. The uniqueness of each labeled target molecule can be determined by the statistics of random choice, and depends on the number of copies of identical target molecules in the collection compared to the diversity of labels. The size of the resulting set of labeled target molecules can be determined by the stochastic nature of the barcoding process, and analysis of the number of barcodes detected then allows calculation of the number of target molecules present in the original collection or sample. When the ratio of the number of copies of a target molecule present to the number of unique barcodes is low, the labeled target molecules are highly unique (i.e., there is a very low probability' that more than one target molecule will have been labeled with a given label).

[0039] As used herein, the term “nucleic acid” refers to a polynucleotide sequence, or fragment thereof. A nucleic acid can comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid can be a gene or fragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA. A nucleic acid can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). Some non-limiting examples of analogs include: 5 -bromouracil, peptide nucleic acid, xenonucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine. and wyosine. “Nucleic acid’; “polynucleotide, “target polynucleotide”, and “target nucleic acid” can be used interchangeably.

[0040] A nucleic acid can comprise one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g.. improved stability). A nucleic acid can comprise a nucleic acid affinity tag. A nucleoside can be a base-sugar combination. The base portion of the nucleoside can be a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides can be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2’, the 3’, or the 5’ hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate groups can covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within nucleic acids, the phosphate groups can commonly be referred to as forming the intemucleoside backbone of the nucleic acid. The linkage or backbone can be a 3’ to 5’ phosphodiester linkage.

[0041] A nucleic acid can comprise a modified backbone and / or modified intemucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones containing a phosphorus atom therein can include, for example, phosphorothioates. chiral phosphorothioates, phosphorodithioates. phosphotri esters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonate such as 3’-alkylene phosphonates, 5’- alkylene phosphonates, chiral phosphonates, phosphinates, phosphorami dates including 3’- amino phosphoramidate and aminoalkyl phosphoramidates. phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates. thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3 ’-5’ linkages, 2’ -5’ linked analogs, and those having inverted polarity wherein one or more intemucleotide linkages is a 3’ to 3’, a 5’ to 5’ or a 2’ to 2’ linkage.

[0042] A nucleic acid can comprise polynucleotide backbones that are formed byshort chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These can include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

[0043] A nucleic acid can comprise a nucleic acid mimetic. The term "‘mimetic’7can be intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the intemucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring can also be referred as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backbone of a polynucleotide can be replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides can be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. The backbone in PNA compounds can comprise two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties can be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

[0044] A nucleic acid can comprise a morpholino backbone structure. For example, a nucleic acid can comprise a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester intemucleoside linkage can replace a phosphodiester linkage.

[0045] A nucleic acid can comprise linked morpholino units (e.g., morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomeric units in a morpholino nucleic acid. Non-ionic morpholino-based oligomeric compounds can have less undesired interactions with cellular proteins. Morpholinobased polynucleotides can be nonionic mimics of nucleic acids. A variety of compounds within the morpholino class can be joined using different linking groups. A further class of polynucleotide mimetic can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a nucleic acid molecule can be replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. The incorporation of CeNA monomers into a nucleic acid chain can increase the stability of a DNA / RNA hybrid. CeNAoligoadenylates can form complexes with nucleic acid complements with similar stability to the native complexes. A further modification can include Locked Nucleic Acids (LNAs) in which the 2’-hydroxyl group is linked to the 4’ carbon atom of the sugar ring thereby forming a 2’-C, 4’-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2). group bridging the 2?oxygen atom and the 4’ carbon atom wherein n is 1 or 2. LNA and LNA analogs can display very high duplex thermal stabilities with complementary nucleic acid (Tm=+3 to +10 °C), stability towards 3’-exonucleolytic degradation and good solubility properties.

[0046] A nucleic acid may also include nucleobase (often referred to simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases can include the purine bases, (e.g., adenine (A) and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine (C) and uracil (U)). Modified nucleobases can include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2 -thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl ( — C=C — CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F- adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified nucleobases can include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin- 2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G- clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pynmido(5,4-(b) (l,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3’,2’:4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0047] As used herein, the term “sample” can refer to a composition comprising targets. Suitable samples for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms.

[0048] As used herein, the term “sampling device” or “device” can refer to a device which may take a section of a sample and / or place the section on a substrate. A sample device can refer to, for example, a fluorescence activated cell sorting (FACS) machine, a cell sortermachine, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and / or a microtome.

[0049] As used herein, the term “solid support” can refer to discrete solid or semisolid surfaces to which a plurality of barcodes (e.g., stochastic barcodes) may be attached. A solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) onto which a nucleic acid may be immobilized (e.g., covalently or non-covalently). A solid support may comprise a discrete particle that may be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. A bead can be non-spherical in shape. A plurality of solid supports spaced in an array may not comprise a substrate. A solid support may be used interchangeably with the term “bead.”

[0050] As used herein, the term “stochastic barcode” can refer to a polynucleotide sequence comprising labels of the present disclosure. A stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcoding. Stochastic barcodes can be used to quantify targets within a sample. Stochastic barcodes can be used to control for errors which may occur after a label is associated with a target. For example, a stochastic barcode can be used to assess amplification or sequencing errors. A stochastic barcode associated with a target can be called a stochastic barcode-target or stochastic barcode-tag-target.

[0051] As used herein, the term “gene-specific stochastic barcode” can refer to a polynucleotide sequence comprising labels and a target-binding region that is gene-specific. A stochastic barcode can be a polynucleotide sequence that can be used for stochastic barcoding. Stochastic barcodes can be used to quantify targets within a sample. Stochastic barcodes can be used to control for errors which may occur after a label is associated with a target. For example, a stochastic barcode can be used to assess amplification or sequencing errors. A stochastic barcode associated with a target can be called a stochastic barcode-target or stochastic barcode- tag-target.

[0052] As used herein, the term “stochastic barcoding” can refer to the random labeling (e.g., barcoding) of nucleic acids. Stochastic barcoding can utilize a recursive Poisson strategy to associate and quantify labels associated with targets. As used herein, the term “stochastic barcoding” can be used interchangeably with “stochastic labeling.”

[0053] As used here, the term “target” can refer to a composition which can be associated with a barcode (e.g., a stochastic barcode). Exemplar}' suitable targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA. and the like. Targets can be single or double stranded. In some embodiments,targets can be proteins, peptides, or polypeptides. In some embodiments, targets are lipids. As used herein, “target” can be used interchangeably with “species.”

[0054] As used herein, the term “reverse transcriptases” can refer to a group of enzymes having reverse transcriptase activity (i.e., that catalyze synthesis of DNA from an RNA template). In general, such enzymes include, but are not limited to. retroviral reverse transcriptase, retrotransposon reverse transcriptase, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptase, and mutants, variants or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transciptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include the Lactococcus lactis LI.LtrB intron reverse transcriptase, the Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or the Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases can include many classes of non-retroviral reverse transcriptases (i.e., retrons, group II introns, and diversity -generating retroelements among others).

[0055] The terms “universal adaptor primer,” “universal primer adaptor” or “universal adaptor sequence” are used interchangeably to refer to a nucleotide sequence that can be used to hybridize to barcodes (e.g., stochastic barcodes) to generate gene-specific barcodes. A universal adaptor sequence can, for example, be a known sequence that is universal across all barcodes used in methods of the disclosure. For example, when multiple targets are being labeled using the methods disclosed herein, each of the target-specific sequences may be linked to the same universal adaptor sequence. In some embodiments, more than one universal adaptor sequences may be used in the methods disclosed herein. For example, when multiple targets are being labeled using the methods disclosed herein, at least two of the target-specific sequences are linked to different universal adaptor sequences. A universal adaptor primer and its complement may be included in two oligonucleotides, one of which comprises a target-specific sequence and the other comprises a barcode. For example, a universal adaptor sequence may be part of an oligonucleotide comprising a target-specific sequence to generate a nucleotide sequence that is complementary to a target nucleic acid. A second oligonucleotide comprising a barcode and a complementary sequence of the universal adaptor sequence may hybridize with the nucleotide sequence and generate a target-specific barcode (e.g.. a target-specific stochastic barcode). In some embodiments, a universal adaptor primer has a sequence that is different from a universal PCR primer used in the methods of this disclosure.Barcodes

[0056] Barcoding, such as stochastic barcoding, has been described in, for example, Fu et al., Proc Natl Acad Sci U.S.A., 2011 May 31,108(22):9026-31; U.S. Patent Application Publication No. US2011 / 0160078; Fan et al., Science, 2015 February 6, 347(6222): 1258367; US Patent Application Publication No. US2015 / 0299784; and PCT Application Publication No. W02015 / 031691; the content of each of these, including any supporting or supplemental information or material, is incorporated herein by reference in its entirety'. In some embodiments, the barcode disclosed herein can be a stochastic barcode which can be a polynucleotide sequence that may be used to stochastically label (e.g., barcode, tag) a target. Barcodes can be referred to stochastic barcodes if the ratio of the number of different barcode sequences of the stochastic barcodes and the number of occurrence of any of the targets to be labeled can be, or be about, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, or a number or a range between any two of these values. A target can be an mRNA species comprising mRNA molecules with identical or nearly identical sequences. Barcodes can be referred to as stochastic barcodes if the ratio of the number of different barcode sequences of the stochastic barcodes and the number of occurrence of any of the targets to be labeled is at least, or is at most, 1 :1, 2: 1, 3: 1, 4: 1, 5:1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15:1. 16:1. 17: 1, 18: 1, 19: 1, 20: 1, 30: 1. 40: 1. 50: 1. 60: 1. 70: 1. 80: 1. 90: 1. or 100: 1. Barcode sequences of stochastic barcodes can be referred to as molecular labels.

[0057] A barcode, for example a stochastic barcode, can comprise one or more labels. Exemplary labels can include a universal label, a cell label, a barcode sequence (e.g., a molecular label), a sample label, a plate label, a spatial label, and / or a pre-spatial label. FIG. 1 illustrates an exemplary barcode 104 with a spatial label. The barcode 104 can comprise a 5 ’amine that may link the barcode to a solid support 105. The barcode can comprise a universal label, a dimension label, a spatial label, a cell label, and / or a molecular label. The order of different labels (including but not limited to the universal label, the dimension label, the spatial label, the cell label, and the molecule label) in the barcode can vary. For example, as shown in FIG. 1, the universal label may be the 5’-most label, and the molecular label may be the 3’-most label. The spatial label, dimension label, and the cell label may be in any order. In some embodiments, the universal label, the spatial label, the dimension label, the cell label, and the molecular label are in any order. The barcode can comprise a target-binding region. The targetbinding region can interact with a target (e.g., target nucleic acid, RNA, mRNA, DNA) in a sample. For example, a target-binding region can comprise an oligo(dT) sequence which can interact with poly(A) tails of mRNAs. In some instances, the labels of the barcode (e.g., universal label, dimension label, spatial label, cell label, and barcode sequence) may beseparated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides.

[0058] A label, for example the cell label, can comprise a unique set of nucleic acid sub-sequences of defined length, e.g.. seven nucleotides each (equivalent to the number of bits used in some Hamming error correction codes), which can be designed to provide error correction capability. The set of error correction sub-sequences comprise seven nucleotide sequences can be designed such that any pairwise combination of sequences in the set exhibits a defined ‘‘genetic distance” (or number of mismatched bases), for example, a set of error correction sub-sequences can be designed to exhibit a genetic distance of three nucleotides. In this case, review of the error correction sequences in the set of sequence data for labeled target nucleic acid molecules (described more fully below) can allow one to detect or correct amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequences used for creating error correction codes can vary’, for example, they can be. or be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50, or a number or a range between any two of these values, nucleotides in length. In some embodiments, nucleic acid sub-sequences of other lengths can be used for creating error correction codes.

[0059] The barcode can comprise a target-binding region. The target-binding region can interact with a target in a sample. The target can be, or comprise, ribonucleic acids (RNAs), messenger RNAs (mRNAs), microRNAs, small interfering RNAs (siRNAs), RNA degradation products, RNAs each comprising a poly(A) tail, or any combination thereof. In some embodiments, the plurality of targets can include deoxyribonucleic acids (DNAs).

[0060] In some embodiments, a target-binding region can comprise an oligo(dT) sequence which can interact with poly(A) tails of mRNAs. One or more of the labels of the barcode (e.g., the universal label, the dimension label, the spatial label, the cell label, and the barcode sequences (e.g., molecular label)) can be separated by a spacer from another one or two of the remaining labels of the barcode. The spacer can be, for example, 1, 2, 3. 4, 5, 6, 7, 8, 9, 10. 11. 12, 13, 14, 15, 16, 17, 18, 19, or 20. or more nucleotides. In some embodiments, none of the labels of the barcode is separated by spacer.Universal Labels

[0061] A barcode can comprise one or more universal labels. In some embodiments, the one or more universal labels can be the same for all barcodes in the set of barcodes attached to a given solid support. In some embodiments, the one or more universal labels can be the same for all barcodes attached to a plurality of beads. In some embodiments, a universal label can comprise a nucleic acid sequence that is capable of hybridizing to a sequencing primer. Sequencing primers can be used for sequencing barcodes comprising a universal label.Sequencing primers (e.g., universal sequencing primers) can comprise sequencing primers associated with high-throughput sequencing platforms. In some embodiments, a universal label can comprise a nucleic acid sequence that is capable of hybridizing to a PCR primer. In some embodiments, the universal label can comprise a nucleic acid sequence that is capable of hybridizing to a sequencing primer and a PCR primer. The nucleic acid sequence of the universal label that is capable of hybridizing to a sequencing or PCR primer can be referred to as a primer binding site. A universal label can comprise a sequence that can be used to initiate transcription of the barcode. A universal label can comprise a sequence that can be used for extension of the barcode or a region within the barcode. A universal label can be, or be about. 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length. For example, a universal label can comprise at least about 10 nucleotides. A universal label can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. In some embodiments, a cleavable linker or modified nucleotide can be part of the universal label sequence to enable the barcode to be cleaved off from the support.Dimension Labels

[0062] A barcode can comprise one or more dimension labels. In some embodiments, a dimension label can comprise a nucleic acid sequence that provides information about a dimension in which the labeling (e.g., stochastic labeling) occurred. For example, a dimension label can provide information about the time at which a target was barcoded. A dimension label can be associated with a time of barcoding (e.g., stochastic barcoding) in a sample. A dimension label can be activated at the time of labeling. Different dimension labels can be activated at different times. The dimension label provides information about the order in which targets, groups of targets, and / or samples were barcoded. For example, a population of cells can be barcoded at the GO phase of the cell cycle. The cells can be pulsed again with barcodes (e.g., stochastic barcodes) at the G1 phase of the cell cycle. The cells can be pulsed again with barcodes at the S phase of the cell cycle, and so on. Barcodes at each pulse (e.g., each phase of the cell cycle), can comprise different dimension labels. In this way, the dimension label provides information about which targets were labelled at which phase of the cell cycle. Dimension labels can interrogate many different biological times. Exemplary' biological times can include, but are not limited to. the cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., a cell, a population of cells) can be labeled before and / or after treatment with a drug and / or therapy. The changes in the number of copies of distinct targets can be indicative of the sample’s response to the drug and / or therapy.

[0063] A dimension label can be activatable. An activatable dimension label can beactivated at a specific time point. The activatable label can be, for example, constitutively activated (e.g., not turned off). The activatable dimension label can be, for example, reversibly activated (e.g., the activatable dimension label can be turned on and turned off). The dimension label can be, for example, reversibly activatable at least 1, 2, 3, 4. 5, 6, 7, 8, 9, 10, or more times. The dimension label can be reversibly activatable, for example, at least 1. 2, 3. 4, 5, 6. 7, 8, 9., 10 or more times. In some embodiments, the dimension label can be activated with fluorescence, light, a chemical event (e.g., cleavage, ligation of another molecule, addition of modifications (e.g., pegylated, sumoylated. acetylated, methylated, deacetylated, demethylated), a photochemical event (e.g., photocaging), and introduction of a non-natural nucleotide.

[0064] The dimension label can, in some embodiments, be identical for all barcodes (e.g., stochastic barcodes) attached to a given solid support (e.g., a bead), but different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%, of barcodes on the same solid support can comprise the same dimension label. In some embodiments, at least 60% of barcodes on the same solid support can comprise the same dimension label. In some embodiments, at least 95% of barcodes on the same solid support can comprise the same dimension label.

[0065] There can be as many as 106or more unique dimension label sequences represented in a plurality of solid supports (e.g.. beads). A dimension label can be. or be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length. A dimension label can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35. 40, 45, 50, 100, 200, or 300, nucleotides in length. A dimension label can comprise between about 5 to about 200 nucleotides. A dimension label can comprise between about 10 to about 150 nucleotides. A dimension label can comprise between about 20 to about 125 nucleotides in length.Spatial Labels

[0066] A barcode can comprise one or more spatial labels. In some embodiments, a spatial label can comprise a nucleic acid sequence that provides information about the spatial orientation of a target molecule which is associated with the barcode. A spatial label can be associated with a coordinate in a sample. The coordinate can be a fixed coordinate. For example, a coordinate can be fixed in reference to a substrate. A spatial label can be in reference to a two or three-dimensional grid. A coordinate can be fixed in reference to a landmark. The landmark can be identifiable in space. A landmark can be a structure which can be imaged. A landmark can be a biological structure, for example an anatomical landmark. A landmark can be a cellular landmark, for instance an organelle. A landmark can be a non-natural landmark such as a structure with an identifiable identifier such as a color code, bar code, magnetic property,fluorescents, radioactivity, or a unique size or shape. A spatial label can be associated with a physical partition (e.g., A well, a container, or a droplet). In some embodiments, multiple spatial labels are used together to encode one or more positions in space.

[0067] The spatial label can be identical for all barcodes attached to a given solid support (e.g., a bead), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes on the same solid support comprising the same spatial label can be, or be about, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or a range between any two of these values. In some embodiments, the percentage of barcodes on the same solid support comprising the same spatial label can be at least, or be at most. 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, at least 60% of barcodes on the same solid support can comprise the same spatial label. In some embodiments, at least 95% of barcodes on the same solid support can comprise the same spatial label.

[0068] There can be as many as 106or more unique spatial label sequences represented in a plurality of solid supports (e.g., beads). A spatial label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length. A spatial label can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200. or 300 nucleotides in length. A spatial label can comprise between about 5 to about 200 nucleotides. A spatial label can comprise between about 10 to about 150 nucleotides. A spatial label can comprise between about 20 to about 125 nucleotides in length.Cell labels

[0069] A barcode (e.g., a stochastic barcode) can comprise one or more cell labels. In some embodiments, a cell label can comprise a nucleic acid sequence that provides information for determining which target nucleic acid originated from which cell. In some embodiments, the cell label is identical for all barcodes attached to a given solid support (e.g., a bead), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes on the same solid support comprising the same cell label can be, or be about 60%, 70%. 80%. 85%. 90%. 95%. 97%. 99%. 100%, or a number or a range between any two of these values. In some embodiments, the percentage of barcodes on the same solid support comprising the same cell label can be, or be about 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least 60% of barcodes on the same solid support can comprise the same cell label. As another example, at least 95% of barcodes on the same solid support can comprise the same cell label.

[0070] There can be as many as 106or more unique cell label sequences represented in a plurality of solid supports (e.g., beads). A cell label can be, or be about, 1. 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotidesin length. A cell label can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. For example, a cell label can comprise between about 5 to about 200 nucleotides. As another example, a cell label can comprise between about 10 to about 150 nucleotides. As yet another example, a cell label can comprise between about 20 to about 125 nucleotides in length.Barcode Sequences

[0071] A barcode can comprise one or more barcode sequences. In some embodiments, a barcode sequence can comprise a nucleic acid sequence that provides identifying information for the specific type of target nucleic acid species hybridized to the barcode. A barcode sequence can comprise a nucleic acid sequence that provides a counter (e.g., that provides a rough approximation) for the specific occurrence of the target nucleic acid species hybridized to the barcode (e.g., target-binding region).

[0072] In some embodiments, a diverse set of barcode sequences are attached to a given solid support (e.g., a bead). In some embodiments, there can be, or be about, 102, 103, 104, 105, 106, 107, 108, 109, or a number or a range between any two of these values, unique molecular label sequences. For example, a plurality7of barcodes can comprise about 6561 barcodes sequences with distinct sequences. As another example, a plurality7of barcodes can comprise about 65536 barcode sequences with distinct sequences. In some embodiments, there can be at least, or be at most, 102, 103. 104, 105, 106, 107, 108, or 109, unique barcode sequences. The unique molecular label sequences can be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular label sequence is partially or entirely encompassed by a particle (e.g.. a hydrogel bead).

[0073] The length of a barcode can be different in different implementations. For example, a barcode can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length. As another example, a barcode can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15. 20. 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.Molecular Labels

[0074] A barcode (e.g., a stochastic barcode) can comprise one or more molecular labels. Molecular labels can include barcode sequences. In some embodiments, a molecular label can comprise a nucleic acid sequence that provides identifying information for the specific type of target nucleic acid species hybridized to the barcode. A molecular label can comprise a nucleic acid sequence that provides a counter for the specific occurrence of the target nucleic acid species hybridized to the barcode (e.g., target-binding region).

[0075] In some embodiments, a diverse set of molecular labels are attached to agiven solid support (e.g., a bead). In some embodiments, there can be, or be about, 102, 103, 104, 105, 106, 107, 108, 109, or a number or a range between any two of these values, of unique molecular label sequences. For example, a plurality of barcodes can comprise about 6561 molecular labels with distinct sequences. As another example, a plurality of barcodes can comprise about 65536 molecular labels with distinct sequences. In some embodiments, there can be at least, or be at most, 102, 103, 104, 105, 106, 107, 108, or 109, unique molecular label sequences. Barcodes with unique molecular label sequences can be attached to a given solid support (e.g., a head).

[0076] For barcoding (e.g., stochastic barcoding) using a plurality of stochastic barcodes, the ratio of the number of different molecular label sequences and the number of occurrence of any of the targets can be, or be about, 1: 1, 2:1, 3: 1, 4: 1, 5: 1, 6:1, 7: 1, 8: 1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1, 100: 1, or a number or a range between any two of these values. A target can be an mRNA species comprising mRNA molecules with identical or nearly identical sequences. In some embodiments, the ratio of the number of different molecular label sequences and the number of occurrence of any of the targets is at least, or is at most, 1: 1, 2:1, 3: 1, 4:1, 5:1, 6: 1, 7: 1, 8:1, 9: 1, 10: 1, 11 : 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1. or 100: 1.

[0077] A molecular label can be, or be about, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or a range between any two of these values, nucleotides in length. A molecular label can be at least, or be at most, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.Target-Bindin Region

[0078] A barcode can comprise one or more target binding regions, such as capture probes. In some embodiments, a target-binding region can hybridize with a target of interest. In some embodiments, the target binding regions can comprise a nucleic acid sequence that hybridizes specifically to a target (e.g., target nucleic acid, target molecule, e.g., a cellular nucleic acid to be analyzed), for example to a specific gene sequence. In some embodiments, a target binding region can comprise a nucleic acid sequence that can attach (e.g., hybridize) to a specific location of a specific target nucleic acid. In some embodiments, the target binding region can comprise a nucleic acid sequence that is capable of specific hybridization to a restriction enzyme site overhang (e.g., an EcoRI sticky-end overhang). The barcode can then ligate to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang.

[0079] In some embodiments, a target binding region can comprise a non-specifictarget nucleic acid sequence. A non-specific target nucleic acid sequence can refer to a sequence that can bind to multiple target nucleic acids, independent of the specific sequence of the target nucleic acid. For example, target binding region can comprise a random multimer sequence, a poly(dA) sequence, a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, or a combination thereof. For example, the target binding region can be an oligo(dT) sequence that hybridizes to the poly(A) tail on mRNA molecules. A random multimer sequence can be, for example, a random dimer, trimer, quatramer, pentamer, hexamer, septamer, octamer, nonamer, decamer, or higher multimer sequence of any length. In some embodiments, the target binding region is the same for all barcodes attached to a given bead. In some embodiments, the target binding regions for the plurality of barcodes attached to a given bead can comprise two or more different target binding sequences. A target binding region can be, or be about, 5, 10, 15, 20, 25, 30, 35, 40. 45, 50, or a number or a range between any two of these values, nucleotides in length. A target binding region can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to generate a cDNA molecule with a poly(dC) tail. A barcode can include a target binding region with a poly(dG) tail. Upon base pairing between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches template strands, from cellular RNA molecule to the barcode, and continues replication to the 5' end of the barcode. By doing so, the resulting cDNA molecule contains the sequence of the barcode (such as the molecular label) on the 3 ’ end of the cDNA molecule.

[0080] In some embodiments, a target-binding region can comprise an oligo(dT) which can hybridize with mRNAs comprising polyadenylated ends. A target-binding region can be gene-specific. For example, a target-binding region can be configured to hybridize to a specific region of a target. A target-binding region can be, or be about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, or a number or a range between any two of these values, nucleotides in length. A target-binding region can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30, nucleotides in length. A target-binding region can be about 5-30 nucleotides in length. When a barcode comprises a gene-specific target-binding region, the barcode can be referred to herein as a gene-specific barcode.Orientation Property

[0081] A stochastic barcode (e.g., a stochastic barcode) can comprise one or more orientation properties which can be used to orient (e.g., align) the barcodes. A barcode can comprise a moiety for isoelectric focusing. Different barcodes can comprise different isoelectricfocusing points. When these barcodes are introduced to a sample, the sample can undergo isoelectric focusing in order to orient the barcodes into a known way. In this way, the orientation property7can be used to develop a known map of barcodes in a sample. Exemplary7orientation properties can include, electrophoretic mobility (e.g., based on size of the barcode), isoelectric point, spin, conductivity, and / or self-assembly. For example, barcodes with an orientation property of self-assembly, can self-assemble into a specific orientation (e g., nucleic acid nanostructure) upon activation.Affinity Property

[0082] A barcode (e.g., a stochastic barcode) can comprise one or more affinityproperties. For example, a spatial label can comprise an affinity7property. An affinity property can include a chemical and / or biological moiety7that can facilitate binding of the barcode to another entity7(e.g., cell receptor). For example, an affinity7property can comprise an antibody, for example, an antibody specific for a specific moiety (e.g.. receptor) on a sample. In some embodiments, the antibody can guide the barcode to a specific cell type or molecule. Targets at and / or near the specific cell type or molecule can be labeled (e.g., stochastically7labeled). The affinity7property7can, in some embodiments, provide spatial information in addition to the nucleotide sequence of the spatial label because the antibody can guide the barcode to a specific location. The antibody can be a therapeutic antibody, for example a monoclonal antibody or a polyclonal antibody. The antibody can be humanized or chimeric. The antibody can be a naked antibody or a fusion antibody.

[0083] The antibody can be a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule, like an antibody7fragment.

[0084] The antibody fragment can be, for example, a portion of an antibody such as F(ab')2, Fab', Fab, Fv, sFv and the like. In some embodiments, the antibody fragment can bind with the same antigen that is recognized by the full-length antibody. The antibody fragment can include isolated fragments consisting of the variable regions of antibodies, such as the “Fv” fragments consisting of the variable regions of the heavy and light chains and recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker (“scFv proteins”). Exemplary antibodies can include, but are not limited to, antibodies for cancer cells, antibodies for viruses, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.Universal Adaptor Primer

[0085] A barcode can comprise one or more universal adaptor primers. For example,a gene-specific barcode, such as a gene-specific stochastic barcode, can comprise a universal adaptor primer. A universal adaptor primer can refer to a nucleotide sequence that is universal across all barcodes. A universal adaptor primer can be used for building gene-specific barcodes. A universal adaptor primer can be, or be about, 1. 2, 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18. 19. 20. 21. 22. 23. 24, 25, 26 27, 28, 29, 30, or a number or a range between any two of these nucleotides in length. A universal adaptor primer can be at least, or be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, or 30 nucleotides in length. A universal adaptor primer can be from 5-30 nucleotides in length.Linker

[0086] When a barcode comprises more than one of a type of label (e.g, more than one cell label or more than one barcode sequence, such as one molecular label), the labels may be interspersed with a linker label sequence. A linker label sequence can be at least about 5, 10, 15, 20, 25, 30. 35. 40. 45, 50 or more nucleotides in length. A linker label sequence can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length. In some instances, a linker label sequence is 12 nucleotides in length. A linker label sequence can be used to facilitate the synthesis of the barcode. The linker label can comprise an error-correcting (e.g., Hamming) code.Solid Supports

[0087] Barcodes, such as stochastic barcodes, disclosed herein can, in some embodiments, be associated with a solid support. The solid support can be, for example, a synthetic particle. In some embodiments, some or all of the barcode sequences, such as molecular labels for stochastic barcodes (e.g., the first barcode sequences) of a plurality of barcodes (e.g., the first plurality of barcodes) on a solid support differ by at least one nucleotide. The cell labels of the barcodes on the same solid support can be the same. The cell labels of the barcodes on different solid supports can differ by at least one nucleotide. For example, first cell labels of a first plurality of barcodes on a first solid support can have the same sequence, and second cell labels of a second plurality of barcodes on a second solid support can have the same sequence. The first cell labels of the first plurality of barcodes on the first solid support and the second cell labels of the second plurality of barcodes on the second solid support can differ by at least one nucleotide. A cell label can be, for example, about 5-20 nucleotides long. A barcode sequence can be, for example, about 5-20 nucleotides long. The synthetic particle can be, for example, a bead.

[0088] The bead can be, for example, a silica gel bead, a controlled pore glass bead, a magnetic bead, a Dynabead, a Sephadex / Sepharose bead, a cellulose bead, a polystyrene bead,or any combination thereof. The bead can comprise a material such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof.

[0089] In some embodiments, the bead can be a polymeric bead, for example a deformable bead or a gel bead, functionalized with barcodes or stochastic barcodes (such as gel beads from 10X Genomics (San Francisco, CA). In some implementation, a gel bead can comprise a polymer based gels. Gel beads can be generated, for example, by encapsulating one or more polymeric precursors into droplets. Upon exposure of the polymeric precursors to an accelerator (e.g., tetramethylethylenediamine (TEMED)), a gel bead may be generated.

[0090] In some embodiments, the particle can be disruptable (e.g., dissolvable, degradable). For example, the polymeric bead can dissolve, melt, or degrade, for example, under a desired condition. The desired condition can include an environmental condition. The desired condition may result in the polymeric bead dissolving, melting, or degrading in a controlled manner. A gel bead may dissolve, melt, or degrade due to a chemical stimulus, a physical stimulus, a biological stimulus, a thermal stimulus, a magnetic stimulus, an electric stimulus, a light stimulus, or any combination thereof.

[0091] Analytes and / or reagents, such as oligonucleotide barcodes, for example, may be coupled / immobilized to the interior surface of a gel bead (e g., the interior accessible via diffusion of an oligonucleotide barcode and / or materials used to generate an oligonucleotide barcode) and / or the outer surface of a gel bead or any other microcapsule described herein. Coupling / immobilization may be via any form of chemical bonding (e.g., covalent bond, ionic bond) or physical phenomena (e.g.. Van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, coupling / immobilization of a reagent to a gel bead or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical cross-linker, including chemical cross-linkers described herein). Upon application of a stimulus, the labile moiety may be cleaved and the immobilized reagent set free. In some embodiments, the labile moiety is a disulfide bond. For example, in the case where an oligonucleotide barcode is immobilized to a gel bead via a disulfide bond, exposure of the disulfide bond to a reducing agent can cleave the disulfide bond and free the oligonucleotide barcode from the bead. The labile moiety may be included as part of a gel bead or microcapsule, as part of a chemical linker that links a reagent or analyte to a gel bead or microcapsule, and / or as part of a reagent or analyte. In some embodiments, at least one barcode of the plurality of barcodes can be immobilized on the particle, partially immobilized on the particle, enclosed in the particle, partially enclosed in the particle, or any combination thereof.

[0092] In some embodiments, a gel bead can comprise a wide range of different polymers including but not limited to: polymers, heat sensitive polymers, photosensitive polymers, magnetic polymers, pH sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. Polymers may include but are not limited to materials such as poly (N -isopropylacrylamide) (PNIPAAm), poly (styrene sulfonate) (PSS), poly (allyl amine) (PAAm), poly(acrylic acid) (PAA), poly(ethylene imine) (PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrolle) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinyl pyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthaladehyde) (PTHF), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).

[0093] Numerous chemical stimuli can be used to trigger the disruption, dissolution, or degradation of the beads. Examples of these chemical changes may include, but are not limited to pH-mediated changes to the bead wall, disintegration of the bead wall via chemical cleavage of crosslink bonds, triggered depolymerization of the bead wall, and bead wall switching reactions. Bulk changes may also be used to trigger disruption of the beads.

[0094] Bulk or physical changes to the microcapsule through various stimuli also offer many advantages in designing capsules to release reagents. Bulk or physical changes occur on a macroscopic scale, in which bead rupture is the result of mechano-physical forces induced by a stimulus. These processes may include, but are not limited to pressure induced rupture, bead wall melting, or changes in the porosity' of the bead wall.

[0095] Biological stimuli may also be used to trigger disruption, dissolution, or degradation of beads. Generally, biological triggers resemble chemical triggers, but many examples use biomolecules, or molecules commonly^ found in living systems such as enzymes, peptides, saccharides, fatty' acids, nucleic acids and the like. For example, beads may comprise polymers with peptide cross-links that are sensitive to cleavage by specific proteases. More specifically, one example may comprise a microcapsule comprising GFLGK peptide cross links. Upon addition of a biological trigger such as the protease Cathepsin B, the peptide cross links of the shell well are cleaved and the contents of the beads are released. In other cases, the proteases may be heat-activated. In another example, beads comprise a shell wall comprising cellulose. Addition of the hydrolytic enzyme chitosan serves as biologic trigger for cleavage of cellulosic bonds, depolymerization of the shell wall, and release of its inner contents.

[0096] The beads may also be induced to release their contents upon the application of a thermal stimulus. A change in temperature can cause a variety changes to the beads. A change in heat may cause melting of a bead such that the bead wall disintegrates. In other cases,the heat may increase the internal pressure of the inner components of the bead such that the bead ruptures or explodes. In still other cases, the heat may transform the bead into a shrunken dehydrated state. The heat may also act upon heat-sensitive polymers within the wall of a bead to cause disruption of the bead.

[0097] Inclusion of magnetic nanoparticles to the bead wall of microcapsules may allow triggered rupture of the beads as well as guide the beads in an array. A device of this disclosure may comprise magnetic beads for either purpose. In one example, incorporation of Fe^O-i nanoparticles into polyelectrolyte containing beads triggers rupture in the presence of an oscillating magnetic field stimulus.

[0098] A bead may also be disrupted, dissolved, or degraded as the result of electrical stimulation. Similar to magnetic particles described in the previous section, electrically sensitive beads can allow for both triggered rupture of the beads as well as other functions such as alignment in an electric field, electrical conductivity or redox reactions. In one example, beads containing electrically sensitive material are aligned in an electric field such that release of inner reagents can be controlled. In other examples, electrical fields may induce redox reactions within the bead wall itself that may increase porosity.

[0099] A light stimulus may also be used to disrupt the beads. Numerous light triggers are possible and may include systems that use various molecules such as nanoparticles and chromophores capable of absorbing photons of specific ranges of wavelengths. For example, metal oxide coatings can be used as capsule triggers. UV irradiation of polyelectrolyte capsules coated with Si O2 may result in disintegration of the bead wall. In yet another example, photo switchable materials such as azobenzene groups may be incorporated in the bead wall. Upon the application of UV or visible light, chemicals such as these undergo a reversible cis-to- trans isomerization upon absorption of photons. In this aspect, incorporation of photon switches result in a bead wall that may disintegrate or become more porous upon the application of a light trigger.

[0100] For example, in a non-limiting example of barcoding (e.g., stochastic barcoding) illustrated in FIG. 2, after introducing cells such as single cells onto a plurality of microwells of a microwell array at block 208, beads can be introduced onto the plurality of microwells of the microwell array at block 212. Each microwell can comprise one bead. The beads can comprise a plurality of barcodes. A barcode can comprise a 5?amine region attached to a bead. The barcode can comprise a universal label, a barcode sequence (e.g., a molecular label), a target-binding region, or any combination thereof.

[0101] The barcodes disclosed herein can be associated with (e.g., attached to) a solid support (e.g., a bead). The barcodes associated with a solid support can each comprise abarcode sequence selected from a group comprising at least 100 or 1000 barcode sequences with unique sequences. In some embodiments, different barcodes associated with a solid support can comprise barcode with different sequences. In some embodiments, a percentage of barcodes associated with a solid support comprises the same cell label. For example, the percentage can be. or be about 60%, 70%, 80%. 85%. 90%. 95%, 97%, 99%, 100%, or a number or a range between any two of these values. As another example, the percentage can be at least, or be at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, barcodes associated with a solid support can have the same cell label. The barcodes associated with different solid supports can have different cell labels selected from a group comprising at least 100 or 1000 cell labels with unique sequences.

[0102] The barcodes disclosed herein can be associated to (e.g., attached to) a solid support (e.g., a bead). In some embodiments, barcoding the plurality of targets in the sample can be performed with a solid support including a plurality of synthetic particles associated with the plurality of barcodes. In some embodiments, the solid support can include a plurality of synthetic particles associated with the plurality of barcodes. The spatial labels of the plurality of barcodes on different solid supports can differ by at least one nucleotide. The solid support can, for example, include the plurality of barcodes in two dimensions or three dimensions. The synthetic particles can be beads. The beads can be silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The solid support can include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the solid supports can be free floating. In some embodiments, the solid supports can be embedded in a semi-solid or solid array. The barcodes may not be associated with solid supports. The barcodes can be individual nucleotides. The barcodes can be associated with a substrate.

[0103] As used herein, the terms “tethered,’' “attached,” and “immobilized,” are used interchangeably, and can refer to covalent or non-covalent means for attaching barcodes to a solid support. Any of a variety of different solid supports can be used as solid supports for attaching pre-synthesized barcodes or for in situ solid-phase synthesis of barcode.

[0104] In some embodiments, the solid support is a bead. The bead can comprise one or more types of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration which a nucleic acid can be immobilized (e.g.. covalently or non-covalently). The bead can be, for example, composed of plastic, ceramic, metal, polymeric material, or any combination thereof. A bead can be, or comprise, a discrete particle that is spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and the like. In some embodiments, a bead can benon-spherical in shape.

[0105] Beads can comprise a variety of materials including, but not limited to, paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (FesCh: magnetite) nanoparticles), ferromagnetic materials (e.g.. iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex. Sepharose, agarose, hydrogel, polymer, cellulose, nylon, or any combination thereof.

[0106] In some embodiments, the bead (e.g., the bead to which the labels are attached) is a hydrogel bead. In some embodiments, the bead comprises hydrogel.

[0107] Some embodiments disclosed herein include one or more particles (for example, beads). Each of the particles can comprise a plurality of oligonucleotides (e.g., barcodes). Each of the plurality of oligonucleotides can comprise a barcode sequence (e.g., a molecular label sequence), a cell label, and a target-binding region (e.g.. an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof). The cell label sequence of each of the plurality of oligonucleotides can be the same. The cell label sequences of oligonucleotides on different particles can be different such that the oligonucleotides on different particles can be identified. The number of different cell label sequences can be different in different implementations. In some embodiments, the number of cell label sequences can be, or be about 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 106, 107, 108, 109, a number or a range between any two of these values, or more. In some embodiments, the number of cell label sequences can be at least, or be at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 106, 107, 108, or 109. In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more of the plurality of the particles include oligonucleotides with the same cell sequence. In some embodiment, the plurality of particles that include oligonucleotides with the same cell sequence can be at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more. In some embodiments, none of the plurality of the particles has the same cell label sequence.

[0108] The plurality of oligonucleotides on each particle can comprise different barcode sequences (e.g., molecular labels). In some embodiments, the number of barcode sequences can be, or be about 10, 100, 200. 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000,80000, 90000, 100000, 106, 107, 108, 109, or a number or a range between any two of these values. In some embodiments, the number of barcode sequences can be at least, or be at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000. 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000. 106, 107, 108, or 109. For example, at least 100 of the plurality of oligonucleotides comprise different barcode sequences. As another example, in a single particle, at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, a number or a range between any two of these values, or more of the plurality of oligonucleotides comprise different barcode sequences. Some embodiments provide a plurality of the particles comprising barcodes. In some embodiments, the ratio of an occurrence (or a copy or a number) of a target to be labeled and the different barcode sequences can be at least 1: 1, 1:2, 1 :3, 1 :4, 1:5, 1 :6, 1:7, 1 :8, 1:9, 1: 10, 1:11, 1 :12, 1 :13, 1 : 14, 1: 15, 1: 16, 1: 17, 1: 18, 1: 19, 1:20, 1:30, 1 :40, 1 :50, 1 :60, 1:70, 1:80, 1 :90, or more. In some embodiments, each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both. The particle can be, for example, a nanoparticle or microparticle.

[0109] The size of the beads can vary. For example, the diameter of the bead can range from 0. 1 micrometer to 50 micrometer. In some embodiments, the diameter of the bead can be, or be about, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometer, or a number or a range between any two of these values.

[0110] The diameter of the bead can be related to the diameter of the wells of the substrate. In some embodiments, the diameter of the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or a range between any two of these values, longer or shorter than the diameter of the well. The diameter of the beads can be related to the diameter of a cell (e.g., a single cell entrapped by a well of the substrate). In some embodiments, the diameter of the bead can be at least, or be at most, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the well. The diameter of the beads can be related to the diameter of a cell (e.g., a single cell entrapped by a well of the substrate). In some embodiments, the diameter of the bead can be, or be about, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or a range between any two of these values, longer or shorter than the diameter of the cell. In some embodiments, the diameter of the beads can be at least, or be at most, 10%, 20%, 30%, 40%, 50%. 60%. 70%, 80%, 90%, 100%, 150%, 200%, 250%. or 300% longer or shorter than the diameter of the cell.[OHl] A bead can be attached to and / or embedded in a substrate. A bead can be attached to and / or embedded in a gel, hydrogel, polymer and / or matrix. The spatial position of a bead within a substrate (e.g.. gel, matrix, scaffold, or polymer) can be identified using the spatiallabel present on the barcode on the bead which can serve as a location address.

[0112] Examples of beads can include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody conjugated beads (e.g., anti-immunoglobulin microbeads), protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorochrome microbeads, and BcMag™ Carboxyl-Terminated Magnetic Beads.

[0113] A bead can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make it fluorescent in one fluorescence optical channel or multiple optical channels. A bead can be associated with iron oxide or chromium oxide to make it paramagnetic or ferromagnetic. Beads can be identifiable. For example, a bead can be imaged using a camera. A bead can have a detectable code associated with the bead. For example, a bead can comprise a barcode. A bead can change size, for example, due to swelling in an organic or inorganic solution. A bead can be hydrophobic. A bead can be hydrophilic. A bead can be biocompatible.

[0114] A solid support (e.g., a bead) can be visualized. The solid support can comprise a visualizing tag (e.g., fluorescent dye). A solid support (e.g., a bead) can be etched with an identifier (e.g.. a number). The identifier can be visualized through imaging the beads.

[0115] A solid support can comprise an insoluble, semi-soluble, or insoluble material. A solid support can be referred to as “functionalized” when it includes a linker, a scaffold, a building block, or other reactive moiety attached thereto, whereas a solid support may be “nonfunctionalized” when it lack such a reactive moiety7attached thereto. The solid support can be employed free in solution, such as in a microtiter well format; in a flow-through format, such as in a column; or in a dipstick.

[0116] The solid support can comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof. A solid support can take the form of resins, gels, microspheres, or other geometric configurations. A solid support can comprise silica chips, microparticles, nanoparticles, plates, arrays, capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold silver, aluminum, silicon and copper), glass supports, plastic supports, silicon supports, chips, filters, membranes, microwell plates, slides, plastic materials including multiwell plates or membranes (e.g., formed of polyethylene, polypropylene, polyamide, polyvinylidenedifluoride), and / or wafers, combs, pins or needles (e.g., arrays of pins suitable for combinatorial synthesis or analysis) or beads in an array of pits or nanoliter wells of flat surfaces such as wafers (e.g., silicon wafers), wafers with pits with or without filter bottoms.

[0117] The solid support can comprise a polymer matrix (e.g., gel. hydrogel). Thepolymer matrix may be able to permeate intracellular space (e.g., around organelles). The polymer matrix may able to be pumped throughout the circulatory system.Substrates and Microwell Array

[0118] As used herein, a substrate can refer to a type of solid support. A substrate can refer to a solid support that can comprise barcodes or stochastic barcodes of the disclosure. A substrate can, for example, comprise a plurality of microwells. For example, a substrate can be a well array comprising two or more microwells. In some embodiments, a microwell can comprise a small reaction chamber of defined volume. In some embodiments, a microwell can entrap one or more cells. In some embodiments, a microwell can entrap only one cell. In some embodiments, a microwell can entrap one or more solid supports. In some embodiments, a microwell can entrap only one solid support. In some embodiments, a microwell entraps a single cell and a single solid support (e.g., a bead). A microwell can comprise barcode reagents of the disclosure.Methods of Barcoding

[0119] The disclosure provides for methods for estimating the number of distinct targets at distinct locations in a physical sample (e.g., tissue, organ, tumor, cell). The methods can comprise placing barcodes (e.g., stochastic barcodes) in close proximity with the sample, lysing the sample, associating distinct targets with the barcodes, amplifying the targets and / or digitally counting the targets. The method can further comprise analyzing and / or visualizing the information obtained from the spatial labels on the barcodes. In some embodiments, a method comprises visualizing the plurality of targets in the sample. Mapping the plurality' of targets onto the map of the sample can include generating a two dimensional map or a three dimensional map of the sample. The two dimensional map and the three dimensional map can be generated prior to or after barcoding (e.g., stochastically barcoding) the plurality of targets in the sample. Visualizing the plurality' of targets in the sample can include mapping the plurality' of targets onto a map of the sample. Mapping the plurality of targets onto the map of the sample can include generating a two dimensional map or a three dimensional map of the sample. The two dimensional map and the three dimensional map can be generated prior to or after barcoding the plurality of targets in the sample, in some embodiments, the two dimensional map and the three dimensional map can be generated before or after lysing the sample. Lysing the sample before or after generating the two dimensional map or the three dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0120] In some embodiments, barcoding the plurality' of targets compriseshybridizing a plurality of barcodes with a plurality of targets to create barcoded targets (e.g., stochastically barcoded targets). Barcoding the plurality of targets can comprise generating an indexed library of the barcoded targets. Generating an indexed library' of the barcoded targets can be performed with a solid support comprising the plurality of barcodes (e.g., stochastic barcodes).Contacting a Sample and a Barcode

[0121] The disclosure provides for methods for contacting a sample (e.g., cells) to a substrate of the disclosure. A sample comprising, for example, a cell, organ, or tissue thin section, can be contacted to barcodes (e.g., stochastic barcodes). The cells can be contacted, for example, by gravity flow wherein the cells can settle and create a monolayer. The sample can be a tissue thin section. The thin section can be placed on the substrate. The sample can be onedimensional (e.g., formsa planar surface). The sample (e.g., cells) can be spread across the substrate, for example, by growing / culturing the cells on the substrate.

[0122] When barcodes are in close proximity to targets, the targets can hybridize to the barcode. The barcodes can be contacted at a non-depletable ratio such that each distinct target can associate with a distinct barcode of the disclosure. To ensure efficient association between the target and the barcode, the targets can be cross-linked to barcode.Cell Lysis

[0123] Following the distribution of cells and barcodes, the cells can be lysed to liberate the target molecules. Cell lysis can be accomplished by any of a variety of means, for example, by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis, or optical lysis. Cells can be lysed by addition of a cell lysis buffer comprising a detergent (e.g., SDS, Li dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or digestive enzymes (e.g., proteinase K, pepsin, or trypsin), or any combination thereof. To increase the association of a target and a barcode, the rate of the diffusion of the target molecules can be altered by for example, reducing the temperature and / or increasing the viscosity of the lysate.

[0124] In some embodiments, the sample can be lysed using a filter paper. The filter paper can be soaked with a lysis buffer on top of the filter paper. The filter paper can be applied to the sample with pressure which can facilitate lysis of the sample and hybridization of the targets of the sample to the substrate.

[0125] In some embodiments, lysis can be performed by mechanical lysis, heat lysis, optical lysis, and / or chemical lysis. Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by the addition of a lysis buffer to the substrate. A lysis buffer can comprise Tris HC1. A lysis buffer can comprise at leastabout 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris HC1. A lysis buffer can comprise at most about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris HCL. A lysis buffer can comprise about 0.1 M Tris HC1. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The pH of the lysis buffer can be at most about 1, 2, 3, 4, 5, 6. 7, 8, 9,10, or more. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer can comprise a salt (e.g., LiCl). The concentration of salt in the lysis buffer can be at least about 0.1, 0.5, or 1 M or more. The concentration of salt in the lysis buffer can be at most about 0.1, 0.5, or 1 M or more. In some embodiments, the concentration of salt in the lysis buffer is about 0.5M. The lysis buffer can comprise a detergent (e.g., SDS, Li dodecyl sulfate, triton X, tween. NP-40). The concentration of the detergent in the lysis buffer can be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or more. The concentration of the detergent in the lysis buffer can be at most about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%. 1%, 2%. 3%, 4%, 5%, 6%, or 7%, or more. In some embodiments, the concentration of the detergent in the lysis buffer is about 1% Li dodecyl sulfate. The time used in the method for lysis can be dependent on the amount of detergent used. In some embodiments, the more detergent used, the less time needed for lysis. The lysis buffer can comprise a chelating agent (e.g., EDTA, EGTA). The concentration of a chelating agent in the lysis buffer can be at least about 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of a chelating agent in the lysis buffer can be at most about 1, 5, 10, 15, 20, 25, or 30mM or more. In some embodiments, the concentration of chelating agent in the lysis buffer is about 10 mM. The lysis buffer can comprise a reducing reagent (e.g., beta-mercaptoethanol. DTT). The concentration of the reducing reagent in the lysis buffer can be at least about 1, 5, 10, 15, or 20 mM or more. The concentration of the reducing reagent in the lysis buffer can be at most about 1, 5, 10, 15, or 20 mM or more. In some embodiments, the concentration of reducing reagent in the lysis buffer is about 5 mM. In some embodiments, a lysis buffer can comprise about 0.1M TrisHCl, about pH 7.5, about 0.5M LiCl, about 1% lithium dodecyl sulfate, about lOmM EDTA, and about 5mM DTT.

[0126] Lysis can be performed at a temperature of about 4, 10, 15, 20, 25, or 30 °C. Lysis can be performed for about 1, 5, 10, 15, or 20 or more minutes. A lysed cell can comprise at least about 100000, 200000, 300000, 400000, 500000, 600000, or 700000 or more target nucleic acid molecules. A lysed cell can comprise at most about 100000, 200000, 300000, 400000, 500000, 600000, or 700000 or more target nucleic acid molecules.Attachment of Barcodes to Target Nucleic Acid Molecules

[0127] Following lysis of the cells and release of nucleic acid molecules therefrom, the nucleic acid molecules can randomly associate with the barcodes of the co-localized solidsupport. Association can comprise hybridization of a barcode’s target recognition region to a complementary portion of the target nucleic acid molecule (e.g., oligo(dT) of the barcode can interact with a poly (A) tail of a target). The assay conditions used for hybridization (e.g., buffer pH, ionic strength, temperature, etc.) can be chosen to promote formation of specific, stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells can associate with the plurality of probes on the substrate (e.g., hybridize with the probes on the substrate). When the probes comprise oligo(dT), mRNA molecules can hybridize to the probes and be reverse transcribed. The oligo(dT) portion of the oligonucleotide can act as a primer for first strand synthesis of the cDNA molecule. For example, in a non-limiting example of barcoding illustrated in FIG. 2, at block 216, mRNA molecules can hybridize to barcodes on beads. For example, single-stranded nucleotide fragments can hybridize to the target-binding regions of barcodes.

[0128] Attachment can further comprise ligation of a barcode’s target recognition region and a portion of the target nucleic acid molecule. For example, the target binding region can comprise a nucleic acid sequence that can be capable of specific hybridization to a restriction site overhang (e.g., an EcoRI sticky-end overhang). The assay procedure can further comprise treating the target nucleic acids with a restriction enzyme (e.g., EcoRI) to create a restriction site overhang. The barcode can then be ligated to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to join the two fragments.

[0129] For example, in a non-limiting example of barcoding illustrated in FIG. 2, at block 220, the labeled targets from a plurality of cells (or a plurality of samples) (e.g., targetbarcode molecules) can be subsequently pooled, for example, into a tube. The labeled targets can be pooled by, for example, retrieving the barcodes and / or the beads to which the targetbarcode molecules are attached.

[0130] The retrieval of solid support-based collections of attached target-barcode molecules can be implemented by use of magnetic beads and an externally-applied magnetic field. Once the target-barcode molecules have been pooled, all further processing can proceed in a single reaction vessel. Further processing can include, for example, reverse transcription reactions, amplification reactions, cleavage reactions, dissociation reactions, and / or nucleic acid extension reactions. Further processing reactions can be performed within the microwells, that is, without first pooling the labeled target nucleic acid molecules from a plurality of cells.Reverse Transcription or Nucleic Acid Extension

[0131] The disclosure provides for a method to create a target-barcode conjugate using reverse transcription (e.g., at block 224 of FIG. 2) or nucleic acid extension. The target-barcode conjugate can comprise the barcode and a complementary sequence of all or a portion of the target nucleic acid (i.e., a barcoded cDNA molecule, such as a stochastically barcoded cDNA molecule). Reverse transcription of the associated RNA molecule can occur by the addition of a reverse transcription primer along with the reverse transcriptase. The reverse transcription primer can be an oligo(dT) primer, a random hexanucleotide primer, or a targetspecific oligonucleotide primer. Oligo(dT) primers can be, or can be about, 12-18 nucleotides in length and bind to the endogenous poly (A) tail at the 3’ end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA at a variety of complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest.

[0132] In some embodiments, reverse transcription of an mRNA molecule to a labeled-RNA molecule can occur by the addition of a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, random hexanucleotide primer, or a target-specific oligonucleotide primer. Generally. oligo(dT) primers are 12-18 nucleotides in length and bind to the endogenous poly (A) tail at the 3’ end of mammalian mRNA. Random hexanucleotide primers can bind to mRNA at a variety of complementary sites. Target-specific oligonucleotide primers ty pically selectively prime the mRNA of interest.

[0133] In some embodiments, a target is a cDNA molecule. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to generate a cDNA molecule with a poly(dC) tail. A barcode can include a target binding region with a poly(dG) tail. Upon base pairing between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches template strands, from cellular RNA molecule to the barcode, and continues replication to the 5' end of the barcode. By doing so, the resulting cDNA molecule contains the sequence of the barcode (such as the molecular label) on the 3’ end of the cDNA molecule.

[0134] Reverse transcription can occur repeatedly to produce multiple labeled-cDNA molecules. The methods disclosed herein can comprise conducting at least about 1, 2. 3, 4, 5. 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The method can comprise conducting at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.Amplification

[0135] One or more nucleic acid amplification reactions (e.g., at block 228 of FIG. 2) can be performed to create multiple copies of the labeled target nucleic acid molecules. Amplification can be performed in a multiplexed manner, wherein multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction can be used to addsequencing adaptors to the nucleic acid molecules. The amplification reactions can comprise amplifying at least a portion of a sample label, if present. The amplification reactions can comprise amplifying at least a portion of the cellular label and / or barcode sequence (e.g., a molecular label). The amplification reactions can comprise amplifying at least a portion of a sample tag, a cell label, a spatial label, a barcode sequence (e.g., a molecular label), a target nucleic acid, or a combination thereof. The amplification reactions can comprise amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a range or a number between any two of these values, of the plurality of nucleic acids. The method can further comprise conducting one or more cDNA synthesis reactions to produce one or more cDNA copies of target-barcode molecules comprising a sample label, a cell label, a spatial label, and / or a barcode sequence (e.g., a molecular label).

[0136] In some embodiments, amplification can be performed using a polymerase chain reaction (PCR). As used herein, PCR can refer to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. As used herein, PCR can encompass derivative forms of the reaction, including but not limited to, RT-PCR. real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR. and assembly PCR.

[0137] Amplification of the labeled nucleic acids can comprise non-PCR based methods. Examples of non-PCR based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA). strand displacement amplification (SDA). real-time SDA, rolling circle amplification, or circle-to-circle amplification. Other non-PCR-based amplification methods include multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify' DNA or RNA targets, a ligase chain reaction (LCR), and a Q(3 replicase (QP) method, use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using a restriction endonuclease, an amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to the extension reaction and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5’ exonuclease activity, rolling circle amplification, and ramification extension amplification (RAM). In some embodiments, the amplification does not produce circularized transcripts.

[0138] In some embodiments, the methods disclosed herein further comprise conducting a polymerase chain reaction on the labeled nucleic acid (e.g., labeled-RNA, labeled- DNA, labeled-cDNA) to produce a labeled amplicon (e.g., a stochastically labeled amplicon).The labeled amplicon can be double-stranded molecule. The double-stranded molecule can comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or a RNA molecule hybridized to a DNA molecule. One or both of the strands of the double-stranded molecule can comprise a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g.. a molecular label). The labeled amplicon can be a single-stranded molecule. The singlestranded molecule can comprise DNA, RNA, or a combination thereof. The nucleic acids of the disclosure can comprise synthetic or altered nucleic acids.

[0139] Amplification can comprise use of one or more non-natural nucleotides. Nonnatural nucleotides can comprise photolabile or triggerable nucleotides. Examples of non-natural nucleotides can include, but are not limited to, peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Non-natural nucleotides can be added to one or more cycles of an amplification reaction. The addition of the non-natural nucleotides can be used to identify products as specific cycles or time points in the amplification reaction.

[0140] Conducting the one or more amplification reactions can comprise the use of one or more primers. The one or more primers can comprise, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers can comprise at least 1, 2, 3, 4. 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers can comprise less than 12-15 nucleotides. The one or more primers can anneal to at least a portion of the plurality of labeled targets (e.g., stochastically labeled targets). The one or more primers can anneal to the 3’ end or 5’ end of the plurality' of labeled targets. The one or more primers can anneal to an internal region of the plurality of labeled targets. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3’ ends the plurality of labeled targets. The one or more primers can comprise a fixed panel of primers. The one or more primers can comprise at least one or more custom primers. The one or more primers can comprise at least one or more control primers. The one or more primers can comprise at least one or more gene-specific primers.

[0141] The one or more primers can comprise a universal primer. The universal primer can anneal to a universal primer binding site. The one or more custom primers can anneal to a first sample label, a second sample label, a spatial label, a cell label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof. The one or more primers can comprise a universal primer and a custom primer. The custom primer can be designed to amplify one or more targets. The targets can comprise a subset of the total nucleic acids in one or moresamples. The targets can comprise a subset of the total labeled targets in one or more samples. The one or more primers can comprise at least 96 or more custom primers. The one or more primers can comprise at least 960 or more custom primers. The one or more primers can comprise at least 9600 or more custom primers. The one or more custom primers can anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids can correspond to one or more genes.

[0142] Any amplification scheme can be used in the methods of the present disclosure. For example, in one scheme, the first round PCR can amplify molecules attached to the bead using a gene specific primer and a primer against the universal Illumina sequencing primer 1 sequence. The second round of PCR can amplify the first PCR products using a nested gene specific primer flanked by Illumina sequencing primer 2 sequence, and a primer against the universal Illumina sequencing primer 1 sequence. The third round of PCR adds P5 and P7 and sample index to turn PCR products into an Illumina sequencing library. Sequencing using 150 bp x 2 sequencing can reveal the cell label and barcode sequence (e.g., molecular label) on read 1, the gene on read 2, and the sample index on index 1 read.

[0143] In some embodiments, nucleic acids can be removed from the substrate using chemical cleavage. For example, a chemical group or a modified base present in a nucleic acid can be used to facilitate its removal from a solid support. For example, an enzyme can be used to remove a nucleic acid from a substrate. For example, a nucleic acid can be removed from a substrate through a restriction endonuclease digestion. For example, treatment of a nucleic acid containing a dUTP or ddUTP with uracil-d-glycosylase (UDG) can be used to remove a nucleic acid from a substrate. For example, a nucleic acid can be removed from a substrate using an enzyme that performs nucleotide excision, such as a base excision repair enzyme, such as an apurinic / apyrimidinic (AP) endonuclease. In some embodiments, a nucleic acid can be removed from a substrate using a photocleavable group and light. In some embodiments, a cleavable linker can be used to remove a nucleic acid from the substrate. For example, the cleavable linker can comprise at least one of biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig-protein A, a photo-labile linker, acid or base labile linker group, or an aptamer.

[0144] When the probes are gene-specific, the molecules can hybridize to the probes and be reverse transcribed and / or amplified. In some embodiments, after the nucleic acid has been synthesized (e.g.. reverse transcribed), it can be amplified. Amplification can be performed in a multiplex manner, wherein multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adaptors to the nucleic acid.

[0145] In some embodiments, amplification can be performed on the substrate, for example, with bridge amplification. cDNAs can be homopolymer tailed in order to generate acompatible end for bridge amplification using oligo(dT) probes on the substrate. In bridge amplification, the primer that is complementary to the 3’ end of the template nucleic acid can be the first primer of each pair that is covalently attached to the solid particle. When a sample containing the template nucleic acid is contacted with the particle and a single thermal cycle is performed, the template molecule can be annealed to the first primer and the first primer is elongated in the forward direction by addition of nucleotides to form a duplex molecule consisting of the template molecule and a newly formed DNA strand that is complementary to the template. In the heating step of the next cycle, the duplex molecule can be denatured, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle through the first primer. In the annealing stage of the annealing and elongation step that follows, the complementary strand can hybridize to the second primer, which is complementary to a segment of the complementary strand at a location removed from the first primer. This hybridization can cause the complementary strand to form a bridge between the first and second primers secured to the first primer by a covalent bond and to the second primer by hybridization. In the elongation stage, the second primer can be elongated in the reverse direction by the addition of nucleotides in the same reaction mixture, thereby converting the bridge to a double-stranded bridge. The next cycle then begins, and the doublestranded bridge can be denatured to yield two single-stranded nucleic acid molecules, each having one end attached to the particle surface via the first and second primers, respectively, with the other end of each unattached. In the annealing and elongation step of this second cycle, each strand can hybridize to a further complementary primer, previously unused, on the same particle, to form new single-strand bridges. The two previously unused primers that are now hybridized elongate to convert the two new bridges to double-strand bridges.

[0146] The amplification reactions can comprise amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of the plurality of nucleic acids.

[0147] Amplification of the labeled nucleic acids can comprise PCR-based methods or non-PCR based methods. Amplification of the labeled nucleic acids can comprise exponential amplification of the labeled nucleic acids. Amplification of the labeled nucleic acids can comprise linear amplification of the labeled nucleic acids. Amplification can be performed by polymerase chain reaction (PCR). PCR can refer to a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. PCR can encompass derivative forms of the reaction, including but not limited to, RT- PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, suppression PCR. semi-suppressive PCR and assembly PCR.

[0148] In some embodiments, amplification of the labeled nucleic acids comprises non-PCR based methods. Examples of non-PCR based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-to-circle amplification. Other non-PCR- based amplification methods include multiple cycles of DNA-dependent RNA polymerase- driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, a ligase chain reaction (LCR), a QP replicase (QP), use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using a restriction endonuclease, an amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to the extension reaction and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5’ exonuclease activity, rolling circle amplification, and / or ramification extension amplification (RAM).

[0149] In some embodiments, the methods disclosed herein further comprise conducting a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon can be double-stranded molecule. The double-stranded molecule can comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or a RNA molecule hybridized to a DNA molecule. One or both of the strands of the double-stranded molecule can comprise a sample tag or molecular identifier label. Alternatively, the amplicon can be a singlestranded molecule. The single-stranded molecule can comprise DNA, RNA, or a combination thereof. The nucleic acids of the present invention can comprise synthetic or altered nucleic acids.

[0150] In some embodiments, the method comprises repeatedly amplifying the labeled nucleic acid to produce multiple amplicons. The methods disclosed herein can comprise conducting at least about 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. or 20 amplification reactions. Alternatively, the method comprises conducting at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0151] Amplification can further comprise adding one or more control nucleic acids to one or more samples comprising a plurality of nucleic acids. Amplification can further comprise adding one or more control nucleic acids to a plurality of nucleic acids. The control nucleic acids can comprise a control label.

[0152] Amplification can comprise use of one or more non-natural nucleotides. Nonnatural nucleotides can comprise photolabile and / or triggerable nucleotides. Examples of nonnatural nucleotides include, but are not limited to, peptide nucleic acid (PNA), morpholino andlocked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Non-natural nucleotides can be added to one or more cycles of an amplification reaction. The addition of the non-natural nucleotides can be used to identify products as specific cycles or time points in the amplification reaction.

[0153] Conducting the one or more amplification reactions can comprise the use of one or more primers. The one or more primers can comprise one or more oligonucleotides. The one or more oligonucleotides can comprise at least about 7-9 nucleotides. The one or more oligonucleotides can comprise less than 12-15 nucleotides. The one or more primers can anneal to at least a portion of the plurality of labeled nucleic acids. The one or more primers can anneal to the 3’ end and / or 5’ end of the plurality of labeled nucleic acids. The one or more primers can anneal to an internal region of the plurality of labeled nucleic acids. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390. 400, 410, 420. 430, 440, 450. 460, 470, 480. 490, 500, 510. 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides from the 3' ends the plurality of labeled nucleic acids. The one or more primers can comprise a fixed panel of primers. The one or more primers can comprise at least one or more custom primers. The one or more primers can comprise at least one or more control primers. The one or more primers can comprise at least one or more housekeeping gene primers. The one or more primers can comprise a universal primer. The universal primer can anneal to a universal primer binding site. The one or more custom primers can anneal to the first sample tag, the second sample tag, the molecular identifier label, the nucleic acid or a product thereof. The one or more primers can comprise a universal primer and a custom primer. The custom primer can be designed to amplify’ one or more target nucleic acids. The target nucleic acids can comprise a subset of the total nucleic acids in one or more samples. In some embodiments, the primers are the probes attached to the array of the disclosure.

[0154] In some embodiments, barcoding (e.g., stochastically barcoding) the plurality’ of targets in the sample further comprises generating an indexed library of the barcoded targets (e.g., stochastically barcoded targets) or barcoded fragments of the targets. The barcode sequences of different barcodes (e.g., the molecular labels of different stochastic barcodes) can be different from one another. Generating an indexed library of the barcoded targets includes generating a plurality of indexed polynucleotides from the plurality’ of targets in the sample. For example, for an indexed library of the barcoded targets comprising a first indexed target and a second indexed target, the label region of the first indexed polynucleotide can differ from the label region of the second indexed polynucleotide by, by about, by at least, or by at most, 1, 2, 3, 4, 5, 6. 7, 8. 9, 10, 20, 30, 40, 50, or a number or a range between any two of these values,nucleotides. In some embodiments, generating an indexed library of the barcoded targets includes contacting a plurality of targets, for example mRNA molecules, with a plurality of oligonucleotides including a poly(T) region and a label region; and conducting a first strand synthesis using a reverse transcriptase to produce single-strand labeled cDNA molecules each comprising a cDNA region and a label region, wherein the plurality of targets includes at least two mRNA molecules of different sequences and the plurality of oligonucleotides includes at least two oligonucleotides of different sequences. Generating an indexed library of the barcoded targets can further comprise amplifying the single-strand labeled cDNA molecules to produce double-strand labeled cDNA molecules: and conducting nested PCR on the double-strand labeled cDNA molecules to produce labeled amplicons. In some embodiments, the method can include generating an adaptor-labeled amplicon.

[0155] Barcoding (e.g., stochastic barcoding) can include using nucleic acid barcodes or tags to label individual nucleic acid (e.g., DNA or RNA) molecules. In some embodiments, it involves adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adaptors can be added for sequencing using, for example, next generation sequencing (NGS). The sequencing results can be used to determine cell labels, molecular labels, and sequences of nucleotide fragments of the one or more copies of the targets, for example at block 232 of FIG. 2.

[0156] FIG. 3 is a schematic illustration showing a non-limiting exemplary process of generating an indexed library of the barcoded targets (e.g., stochastically barcoded targets), such as barcoded mRNAs or fragments thereof. As shown in step 1. the reverse transcription process can encode each mRNA molecule with a unique molecular label sequence, a cell label sequence, and a universal PCR site. In particular, RNA molecules 302 can be reverse transcribed to produce labeled cDNA molecules 304, including a cDNA region 306, by hybridization (e.g., stochastic hybridization) of a set of barcodes (e.g., stochastic barcodes) 310 to the poly(A) tail region 308 of the RNA molecules 302. Each of the barcodes 310 can comprise a target-binding region, for example a poly(dT) region 312, a label region 314 (e.g., a barcode sequence or a molecule), and a universal PCR region 316.

[0157] In some embodiments, the cell label sequence can include 3 to 20 nucleotides. In some embodiments, the molecular label sequence can include 3 to 20 nucleotides. In some embodiments, each of the plurality of stochastic barcodes further comprises one or more of a universal label and a cell label, wherein universal labels are the same for the plurality of stochastic barcodes on the solid support and cell labels are the same for the plurality7of stochastic barcodes on the solid support. In some embodiments, the universal label can include 3to 20 nucleotides. In some embodiments, the cell label comprises 3 to 20 nucleotides.

[0158] In some embodiments, the label region 314 can include a barcode sequence or a molecular label 318 and a cell label 320. In some embodiments, the label region 314 can include one or more of a universal label, a dimension label, and a cell label. The barcode sequence or molecular label 318 can be, can be about, can be at least, or can be at most, 1, 2. 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length. The cell label 320 can be, can be about, can be at least, or can be at most, 1, 2, 3, 4, 5, 6, 7, 8. 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length. The universal label can be, can be about, can be at least, or can be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length. Universal labels can be the same for the plurality of stochastic barcodes on the solid support and cell labels are the same for the plurality of stochastic barcodes on the solid support. The dimension label can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length.

[0159] In some embodiments, the label region 314 can comprise, comprise about, comprise at least, or comprise at most, 1, 2. 3, 4, 5. 6, 7, 8. 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500. 600, 700, 800. 900, 1000, or a number or a range between any of these values, different labels, such as a barcode sequence or a molecular label 318 and a cell label 320. Each label can be, can be about, can be at least, or can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or a range between any of these values, of nucleotides in length. A set of barcodes or stochastic barcodes 310 can contain, contain about, contain at least, or can be at most, 10, 20, 40, 50, 70, 80, 90, 102, 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 102°, or a number or a range between any of these values, barcodes or stochastic barcodes 310. And the set of barcodes or stochastic barcodes 310 can, for example, each contain a unique label region 314. The labeled cDNA molecules 304 can be purified to remove excess barcodes or stochastic barcodes 310. Purification can comprise Ampure bead purification.

[0160] As shown in step 2, products from the reverse transcription process in step 1 can be pooled into 1 tube and PCR amplified with a 1stPCR primer pool and a 1stuniversal PCR primer. Pooling is possible because of the unique label region 314. In particular, the labeled cDNA molecules 304 can be amplified to produce nested PCR labeled amplicons 322. Amplification can comprise multiplex PCR amplification. Amplification can comprise a multiplex PCR amplification with 96 multiplex primers in a single reaction volume. In some embodiments, multiplex PCR amplification can utilize, utilize about, utilize at least, or utilize atmost, 10, 20, 40, 50, 70, 80, 90, 102, 103, 104, IO3, 106, 107, 108, IO9, IO10, IO11, 1012, IO13, IO14, 1013, IO20, or a number or a range between any of these values, multiplex primers in a single reaction volume. Amplification can comprise using a 1stPCR primer pool 324 comprising custom primers 326A-C targeting specific genes and a universal primer 328. The custom primers 326 can hybridize to a region within the cDNA portion 306’ of the labeled cDNA molecule 304. The universal primer 328 can hybridize to the universal PCR region 316 of the labeled cDNA molecule 304.

[0161] As shown in step 3 of FIG. 3, products from PCR amplification in step 2 can be amplified with a nested PCR primers pool and a 2nduniversal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, the nested PCR labeled amplicons 322 can be further amplified by nested PCR. The nested PCR can comprise multiplex PCR with nested PCR primers pool 330 of nested PCR primers 332a-c and a 2nduniversal PCR primer 328’ in a single reaction volume. The nested PCR primer pool 328 can contain, contain about, contain at least, or contain at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or a range between any of these values, different nested PCR primers 330. The nested PCR primers 332 can contain an adaptor 334 and hybridize to a region within the cDNA portion 306" of the labeled amplicon 322. The universal primer 328’ can contain an adaptor 336 and hybridize to the universal PCR region 316 of the labeled amplicon 322. Thus, step 3 produces adaptor-labeled amplicon 338. In some embodiments, nested PCR primers 332 and the 2nduniversal PCR primer 328’ may not contain the adaptors 334 and 336. The adaptors 334 and 336 can instead be ligated to the products of nested PCR to produce adaptor-labeled amplicon 338.

[0162] As shown in step 4, PCR products from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, the adaptors 334 and 336 can be used to conduct one or more additional assays on the adaptor-labeled amplicon 338. The adaptors 334 and 336 can be hybridized to primers 340 and 342. The one or more primers 340 and 342 can be PCR amplification primers. The one or more primers 340 and 342 can be sequencing primers. The one or more adaptors 334 and 336 can be used for further amplification of the adaptor-labeled amplicons 338. The one or more adaptors 334 and 336 can be used for sequencing the adaptor-labeled amplicon 338. The primer 342 can contain a plate index 344 so that amplicons generated using the same set of barcodes or stochastic barcodes 310 can be sequenced in one sequencing reaction using next generation sequencing (NGS).In situ RNA probing

[0163] mRNA from formalin fixed cells are cross-linked and it is not easy for thesemolecules to released and captured by barcoding particles (e.g., Rhapsody beads). To address this outstanding issue, in some embodiments provided herein RNA probes are provided which can bind to said mRNAs and in situ reverse transcription can generate short cDNA connected to the probes. These probes can be easily captured by barcoding particles (e.g., Rhapsody beads) and the use of specific targeted panel primers can improve specificity and eliminate potential non-specificity of probes. Targeted probe panels and primer panels are provided herein, as well as in situ probe hybridization and RT kits. There are provided, in some embodiments, methods and compositions for RNA probing and amplification on single cell analysis systems (e.g., Rhapsody) to expand sample types to fixed (e.g., formalin-fixed) cells. Methods and compositions disclosed herein enable single cell transcriptome profiling of single cells from fixed samples via an RNA probing assay and single cell analysis systems (e.g., Rhapsody). In some embodiments current single cell analysis workflows (e.g.. Rhapsody) are unable to provide gene expression profiling solutions on cells from formaline-based fixatives since mRNAs are cross-linked and not available to capture by conventional mRNA capture methods (e.g., via poly A / dT capture). In some embodiments, and without being bound by any particular theory, although it is hard to release the mRNA from fixed cells, small DNA probes can enter the fixed cells and bind to the target mRNAs, in methods such as, for example, in situ hybridization or FISH, to visualize mRNAs. However, currently the number of probes that can be used to detect mRNAs are limited due to the number of fluorescent detectors and it is thus difficult to provide transcriptome solutions with these approaches. In some embodiments of the compositions and methods provided herein, mRNA sequence-specific probes can be used to bind to target mRNAs and can be extended by in situ reverse transcription to gain the sequence from the mRNA to gain another layer of specificity. Moreover, in some embodiments disclosed herein, a high number target panel can be designed without limitation of the detection method. After probing / in situ RT, cells can be washed to remove un-bound probes and loaded onto single cell analysis platforms (e.g., Rhapsody). Probes with cDNA attached can be released from single cells and can captured by oligonucleotide barcodes associated with barcoding particles (e.g., Rhapsody beads) through a TSO capture oligo via a capture sequence added onto the probe. Then a user can employ a targeted primer panel to amplify targeted genes from the beads alongside of cell label and UMI to analyze single cell targeted mRNA profiling via sequencing. The disclosed compositions and methods can open up the usage of archived formaline-fixed cells for single cell RNAseq analysis. Additionally, there are provided, in some embodiments, methods and compositions enabling spatial gene expression studies on FFPE fixed tissue sections. Samples which can be used in current single cell analysis systems (e.g., Rhapsody) are limited to live cells, fresh isolated nuclei or short-time preserved cells. The disclosed compositions andmethods can enable the use of long-term stored formalin-fixed samples on single cell analysis systems (e.g., Rhapsody).

[0164] FIGS. 4A-4D depict a non-limiting exemplary' schematic workflow for gene expression analysis of fixed cells. The workflow can comprise contacting a sample (e.g., a fixed sample comprising fixed cell(s)) with a plurality of probing oligonucleotides (step 400a). Each of the probing oligonucleotides can comprise a coupling sequence and a probe sequence configured hybridize a nucleic acid target within the sample. The 5’ end of each probing oligonucleotide can be phosphorylated. The probing oligonucleotides can be capable of entering a cell and / or a nucleus of the sample (e.g., a permeabilized cell and / or a permeabilized nucleus of the sample). The workflow can comprise after contacting the probing oligonucleotides with the sample, removing one or more probing oligonucleotides of the plurality of probing oligonucleotides that are not contacted with the sample. Removing the one or more probing oligonucleotides not contacted with the sample can comprise removing the one or more probing oligonucleotides that have not entered a cell of the sample. The workflow can comprise extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary' to at least a portion of the nucleic acid target (step 400b). The workflow can comprise contacting the sample with extension reagents. The extension reagents can comprise reverse transcription reagents (e g., a reverse transcriptase and dNTPs). The extension can be performed in situ and can comprise in situ reverse transcription. In some embodiments, cells of the sample remain intact during the extension step. The sample can comprise a plurality of cells, and the workflow can comprises disassociating the sample to generate a plurality of single cells. The workflow can comprise partitioning the plurality' of single cells to a plurality' of partitions. The workflow can comprise contacting the plurality' of extended probing oligonucleotides with a barcoding particle (e.g., Rhapsody bead) (step 400c). The barcoding particle (e.g., bead) can be associated with a plurality of oligonucleotide barcodes comprising one or more of a cell label (CL), molecular lable (UMI), a 5’ first universal sequence, and a 3?TSO (e.g.. capture sequence). The workflow can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides. Barcoding the plurality of extended probing oligonucleotides can comprise: providing a splint oligonucleotide (e.g., coupling oligonucleotide) comprising a 5’ complement of the coupling sequence and a 3’ complement of a capture sequence; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5‘ complement of the coupling sequence of the coupling oligonucleotide; hybridizing the 3’ complement of the capture sequence of the coupling oligonucleotide with a capturesequence of an oligonucleotide barcode of the plurality of oligonucleotide barcodes; and / or ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode. The workflow can comprise amplifying the plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or complement thereof, and amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, thereby generating a plurality of amplified barcoded probing oligonucleotides (step 400d). The amplification primer(s) can comprise a second universal sequence (e.g., R2 primer sequence) and / or the first primer can comprise a third universal sequence. The workflow can comprise obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides, or products thereof (step 400e). Obtaining sequencing data can comprise attaching the binding sites of sequencing primers and / or sequencing adaptors to the plurality of barcoded probing oligonucleotides, or products thereof. The workflow can comprise determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of amplified barcoded probing oligonucleotides, or products thereof. In some embodiments the probing oligonucleotides comprise a predetermined spatial label, and the workflow comprises determining the spatial location and copy number of a nucleic acid target in the sample.

[0165] Some embodiments provide methods for labeling nucleic acid targets in a sample. In some embodiments, the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target. The method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides,or products thereof.

[0166] Some embodiments provide methods for determining the copy number of a nucleic acid target in a sample. In some embodiments, the method comprises: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality' of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target. The method can comprise: determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.

[0167] Some embodiments provide methods for determining the spatial location and copy number of a nucleic acid target in a sample. In some embodiments, the method comprises: contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and a predetermined spatial label. In some embodiments probing oligonucleotides contacted with the same spatial location comprise the same spatial label sequence, and wherein probing oligonucleotides contacted with distinct spatial locations of the sample comprise different spatial label sequences. The method can comprise: extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary’ to at least a portion of the nucleic acid target. The method can comprise: barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of theplurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target. The method can comprise: obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target. The method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a nucleic acid target to determine the copy number of the nucleic acid target at each spatial location of the sample.

[0168] The method can comprise: contacting the sample with extension reagents. At least a portion of the contacting step can be performed in the presence in the presence of extension reagents. The entire contacting step can be performed in the presence in the presence of the extension reagents. The contacting and extension steps can be simultaneous. The extension can be performed in situ. Said extension can comprise in situ reverse transcription. In some embodiments, cells of the sample remain intact during the extension step. The extension reagents can comprise reverse transcription reagents. Reverse transcription reagents can comprise a reverse transcriptase and dNTPs. The reverse transcriptase can comprise a viral reverse transcriptase. The viral reverse transcriptase can be a murine leukemia virus (MLV) reverse transcriptase or a Moloney murine leukemia virus (MMLV) reverse transcriptase.

[0169] Barcoding the plurality of extended probing oligonucleotides, or products thereof, can comprise: providing a coupling oligonucleotide comprising a 5' complement of the coupling sequence and a 3’ complement of a capture sequence; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5’ complement of the coupling sequence of the coupling oligonucleotide; hybridizing the 3’ complement of the capture sequence of the coupling oligonucleotide with a capture sequence of an oligonucleotide barcode of the plurality of oligonucleotide barcodes; and / or ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode. The method can comprise: before ligating the extended probing oligonucleotide to the oligonucleotide barcode, filling a gap between the extended probing oligonucleotide and the hybridized oligonucleotide barcode with a DNA polymerase lacking at least one of 5 ' to 3’ exonuclease activity and 3’ to 5’ exonuclease activity. Ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode can be performed with a DNA ligase. The coupling oligonucleotide can be a singlestranded oligonucleotide, a double-stranded oligonucleotide, or a mixture thereof. The coupling oligonucleotide can comprise non-natural nucleotides. The coupling oligonucleotide can comprise at least about 1, 2, 3, 4, 5. 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides. The coupling sequence can comprise at least about 1, 2. 3, 4, 5. 6. 7, 8, 9. 10. 11. 12. 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84. 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, or a number or a range between any two of these values, nucleotides. The 5’ end of each probing oligonucleotide can be phosphorylated. The probing oligonucleotides can be capable of entering a cell and / or a nucleus of the sample (e.g., a permeabilized cell and / or a permeabilized nucleus of the sample). The method can comprise: after contacting the probing oligonucleotides with the sample, removing one or more probing oligonucleotides of the plurality of probing oligonucleotides that are not contacted with the sample, optionally removing the one or more probing oligonucleotides not contacted with the sample comprises: removing the one or more probing oligonucleotides that have not entered a cell of the sample.

[0170] The contacting step can comprise contacting the sample with a device configured to deposit probing oligonucleotides (e.g., an ink jet device). The device can be a needle, a needle array, a tube, a suction device, an injection device, an electroporation device, a fluorescent activated cell sorter device, an ink jet device, a microfluidic device, or any combination thereof. In some embodiments, the device contacts distinct spatial locations of the sample at a specified rate. The spatial label can be at least about 1, 2, 3, 4, 5, 6, 7, 8. 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93. 94, 95, 96, 97, 98, 99, 100, or a number or a range between any two of these values, nucleotides in length. Said two or more spatial locations can comprise at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, distinct spatial locations of the sample. In some embodiments, a spatial location of the sample corresponds to an area comprising no more than about 50 cells, about 45 cells, about 40 cells, about 35 cells, about 30 cells, about 25 cells, about 20 cells, about 15 cells, about 10 cells, about 9 cells, about 8 cells, about 7 cells, about 6 cells, about 5 cells, about 4 cells, about 3 cells, about 2 cells, about 1 cell, or a number or a range between any two of these values.

[0171] Each oligonucleotide barcode of the plurality of oligonucleotide barcodes cancomprise a first universal sequence. In some embodiments, obtaining sequencing data comprises: amplifying the plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or complement thereof, and amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, thereby generating a plurality of amplified barcoded probing oligonucleotides. Obtaining sequencing data can comprise obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides, or products thereof. Obtaining sequencing data can comprise attaching the binding sites of sequencing primers and / or sequencing adaptors to the plurality of barcoded probing oligonucleotides, or products thereof. The amplification primer(s) can comprise a second universal sequence and / or the first primer can comprise a third universal sequence. The first universal sequence, the second universal sequence, and / or the third universal sequence can be the same. The first universal sequence, the second universal sequence, and / or the third universal sequence can be different. The first universal sequence, the second universal sequence, and / or the third universal sequence can comprise the binding sites of sequencing primers and / or sequencing adaptors, complementary sequences thereof, and / or portions thereof. The sequencing adaptors can comprise a P5 sequence, a P7 sequence, complementary' sequences thereof, and / or portions thereof. The sequencing primers can comprise a Read 1 sequencing primer, a Read 2 sequencing primer, complementary sequences thereof, and / or portions thereof.

[0172] The sample can comprise a plurality of nucleic acid targets, such as, for example, a target panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12. about 14, about 16, about 18. about 20, about 30, about 40. about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, or a number or a range between any two of these values, distinct nucleic acid targets. Two or more nucleic acid targets of the target panel can be biomarkers. The biomarkers can be biomarkers for a disease or condition. The disease or condition can be a cancer, an infection, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a bacterial infection, or any combination thereof. The contacting step can comprise contacting the sample with a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality comprises a probe sequence configured hybridize a nucleic acid target of the plurality of nucleic acid targets. Determining the copy number of the nucleic acid target in the sample can comprise determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of molecular labels with distinct sequences associated with the plurality ofbarcoded probing oligonucleotides, or products thereof, comprising a sequence of the each of the plurality of nucleic acid targets. The method can comprise: for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with each of the plurality of nucleic acid targets to determine the copy number of each of the plurality of nucleic acid targets at each spatial location of the sample. The amplification primer(s) can comprise a panel of amplification primers configured to hybridize the plurality of nucleic acid targets, or complements thereof, such as, for example, a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9. about 10, about 12, about 14, about 16, about 18, about 20. about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, or a number or a range between any two of these values, distinct amplification primers. As used herein, the term ’panel" shall be given its ordinary meaning and shall also refer to a set of nucleic acids designed to hybridize a set of target nucleic acid sequences of interest, or products thereof. For example, in some embodiments, expression analysis of 200 genes can be performed using a panel of 200 different probing oligonucleotides (e.g., a panel of probing oligonucleotides comprising 200 pluralities of probing oligonucleotides) designed to bind the 200 transcripts of said genes. In some such embodiments, following extension of these probing oligonucleotides in situ, the extension products (extended probing oligonucleotides) can be barcoded as described herein and then amplified using a panel of 200 different amplification primers designed to bind a sequence of the 200 nucleic acid targets (or complements thereof). The nucleic acid target can comprise a nucleic acid molecule (e.g., a ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation product, RNA comprising a poly(A) tail, a sample indexing oligonucleotide, a cellular component-binding reagent specific oligonucleotide, or any combination thereof).

[0173] Each molecular label of the plurality of oligonucleotide barcodes can comprise at least 6 nucleotides. Each capture sequence of the plurality of oligonucleotide barcodes can comprise at least 4 nucleotides. The plurality of oligonucleotide barcodes can be associated with a solid support, and a partition of the plurality' of partitions can comprise a single solid support. The plurality of oligonucleotide barcodes each can comprise a cell label. Each cell label of the plurality of oligonucleotide barcodes can comprise at least 6 nucleotides. Oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with the same solid support can comprise the same cell label. Oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with different solid supports can comprise different celllabels. The solid support can comprise a synthetic particle, a planar surface, or a combination thereof. The method can comprise: associating a synthetic particle comprising the plurality of oligonucleotide barcodes with the cell in the partition. The method can comprise: lysing the cell after associating the synthetic particle with the cell. Lysing the cell can comprise heating the cell, contacting the cell with a detergent, changing the pH of the cell, or any combination thereof. The synthetic particle and the single cell can be in the same partition, and the partition can be a well or a droplet. At least one oligonucleotide barcode of the plurality' of oligonucleotide barcodes can be immobilized or partially immobilized on the synthetic particle, and / or at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes can be enclosed or partially enclosed in the synthetic particle. The synthetic particle can be disruptable (e.g., a disruptable hydrogel particle). The synthetic particle can comprise a bead. The bead can comprise a Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A / G conjugated bead, a protein L conjugated bead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, or any combination thereof. The synthetic particle can comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. Each oligonucleotide barcode of the plurality of oligonucleotide barcodes can comprise a linker functional group. The synthetic particle can comprise a solid support functional group. The support functional group and the linker functional group can be associated with each other, and the linker functional group and the support functional group can be individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s), and any combination thereof.Sample Analysis

[0174] The sample can be physically divided or can be intact during the contacting step. The sample can comprise a single cell. The sample can comprise a plurality of single cells. The sample can comprise a plurality of cells, and the method can comprises: disassociating the sample to generate a plurality of single cells. Said disassociating can comprise chemical dissociating, enzymatic dissociating, and / or mechanical dissociating. Said disassociating can employ one or more of collagenase, chymotrypsin, dispase, elastase, hyaluronidase, pancreatin, papain, and trypsin. The method can comprise: prior to the barcoding step: partitioning the plurality of single cells to a plurality7of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality7of single cells; and in the partition comprising the single cell, contacting the extended probing oligonucleotide with the plurality ofoligonucleotide barcodes. In some embodiments, the method comprises: in the partition comprising the single cell, contacting the single cell with a lysis buffer at 15-65 °C to lyse the single cell. The lysis buffer can comprise an agent capable of dissociating protein-nucleic acid complexes.

[0175] The plurality of cells can comprise one or more cell types. Said one or more cell types can be selected from the group consisting of: brain cells, heart cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary' cells, and circulatory cells, or any combination thereof. The sample can comprise a biological sample, a clinical sample, an environmental sample, a biological fluid, a tissue, a tissue section derived from a subject, or any combination thereof. The subject can be a human, a mouse, a dog, a rat, or a vertebrate. The method can comprise: determining genoty pe, phenotype, or one or more genetic mutations of the subject based on the spatial location of the nucleic acid targets in the sample. The method can comprise: predicting susceptibility of the subject to one or more diseases, such as, for example, a cancer or a hereditary disease. The method can comprise: determining cell ty pes of the plurality7of cells in the sample. In some embodiments, a drug can be chosen based on predicted responsiveness of the cell ty pes of the plurality7of cells in the sample.

[0176] The method can comprise imaging the sample, optionally imaging the sample before the contacting step and / or after the contacting step, optionally the imaging generates imaging data. Imaging the sample can comprise staining the sample with a stain, the stain can be a fluorescent stain, a negative stain, an antibody stain, or any combination thereof. Staining can comprise Immunocytochemistry (ICC), Immunohistochemistry (IHC), Immunofluorescence (IF), or any combination thereof. In some embodiments, imaging can comprise microscopy, confocal microscopy, time-lapse imaging microscopy, fluorescence microscopy, multi-photon microscopy, quantitative phase microscopy, surface enhanced Raman spectroscopy, videography, manual visual analysis, automated visual analysis, or any combination thereof. The method can comprise: associating the imaging data and sequencing data of one or more spatial locations of the sample. The method can comprise: correlation analysis of the imaging data and the sequencing data of the spatial locations. The correlation analysis can identify one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of therapeutic agents, and / or cellular targets of candidate therapeutic agents. In some embodiments, said imaging produces an image that is used to construct a map of a physical representation of said sample. In some embodiments, said map can be two dimensional or three dimensional. The method can comprise: mapping the nucleic acid targets and / or cellular component targets onto the map of the sample. The method can comprise: mapping one or more single cells of theplurality of cells onto the map of the sample. Sequencing reads derived from the same single cell of the plurality of cells can comprise the same cell label. These sequence reads can also comprise the sequence of the spatial label. A user can associate a single cell of the sample with a spatial location of the sample based on the association of the cell label and the spatial label.Cellular Component Target Profiling

[0177] The sample can comprise a plurality of cellular component targets, and the method can comprises: contacting a plurality of cellular component-binding reagents with the sample, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular componentbinding reagent is capable of specifically binding to at least one of the plurality of cellular component targets; barcoding the cellular component-binding reagent specific oligonucleotides to generate a plurality of barcoded cellular component-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique identifier sequence and a molecular label sequence; and obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and at least a portion of the unique identifier sequence. Obtaining sequencing data can comprise attaching the binding sites of sequencing primers and / or sequencing adaptors to the barcoded cellular component-binding reagent specific oligonucleotides, or products thereof.

[0178] The method can comprise: after contacting the plurality of cellular component-binding reagents with the sample, removing one or more cellular component-binding reagents of the plurality of cellular component-binding reagents that are not contacted with the sample, optionally removing the one or more cellular component-binding reagents not contacted with the sample comprises: removing the one or more cellular component-binding reagents not contacted with the respective at least one of the plurality’ of cellular component targets. The cellular component target can comprise an intracellular protein, a carbohydrate, a lipid, a protein, an extracellular protein, a cell-surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof. The cellular component-binding reagent specific oligonucleotide can comprise a second molecular label, optionally at least ten of the plurality of cellular component-binding reagent specific oligonucleotides comprise different second molecular label sequences. The second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides can be different, and the unique identifiersequences of the at least two cellular component-binding reagent specific oligonucleotides can be identical. The second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides can be different, and the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides can be different. In some embodiments, the number of unique molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample. In some embodiments, the number of unique second molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample.

[0179] In some embodiments, cellular component-binding reagent specific oligonucleotides are barcoded using the same plurality of oligonucleotides barcodes as used to barcode the extended probing oligonucleotides. In some embodiments the solid support comprises two or more pluralities of oligonucleotide barcodes wherein each plurality comprises a distinct 3’ target-binding region or capture sequence. For example, in some embodiments described herein, the extended probing oligonucleotides are barcoded with a first plurality of oligonucleotides barcodes having a 3’ capture sequence configured to hybridize to a coupling oligonucleotide (e.g., TSO bait) and cellular component-binding reagent specific oligonucleotides are barcoded using a second plurality of oligonucleotide barcodes having a 3’ poly(dT) sequence. In some such embodiments, the cellular component-binding reagent specific oligonucleotides comprise a poly(dA) sequence.

[0180] There are provided, in some embodiments, methods for determining the spatial location and copy number of a cellular component target in a sample. Sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof, can each comprise a cell label sequence. As described above, a user can associate a single cell of the sample with a spatial location of the sample based on the association of the cell label and the spatial label. Accordingly, a user can thereby determine the spatial location and copy number of a cellular component target in a sample based on the spatial label (and thereby spatial location) associated with said cell label in the sequencing data.

[0181] Embodiments of using cellular component binding reagents (e.g., proteinbinding regents) that are associated with oligonucleotides (for example, oligo-conjugated antibodies (AbOs) and oligo-conjugated aptamers) for barcoding and / or for determining protein expression profiles in single cells and sample tracking (e.g., tracking sample origins) have beendescribed in US2018 / 0088112 and US2018 / 0346970; and WG / 2020 / 037065; the content of each of these applications is incorporated herein by reference in its entirety. The systems, methods, compositions, and kits provided herein can, in some embodiments, be employed in concert with the systems, methods, compositions, and kits described in PCT Application Publication No. WO / 2021 / 163374. the content of which is incorporated herein by reference in its entirety. The systems, methods, compositions, and kits provided herein can, in some embodiments, be employed in concert with the systems, methods, compositions, and kits described in PCT Application Publication Nos. WO / 2024 / 097719 and WO / 2024 / 097718; the content of each of these applications is incorporated herein by reference in its entirety.Fixing Agents, Unfixing Agents, and Permeabilizing Agents

[0182] The sample can comprise a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof. The sample can comprise a fresh tissue section, a frozen tissue section, a fixed tissue section, a formalin-fixed tissue section, a formalin-fixed paraffin- embedded (FFPE) tissue section, an acetone fixed tissue section, a paraformaldehyde (PFA) fixed tissue section, and / or a methanol fixed tissue section. The sample can comprise a nuclei suspension, such as, for example, a fixed nuclei suspension and / or a permeabilized nuclei suspension. In some embodiments, the sample has been contacted with one or more fixing agents and / or permeabilizing agents. The sample can comprise cell(s), such as. for example, fresh cell(s), frozen cell(s), fixed cell(s), formalin-fixed cell(s), formalin-fixed paraffin- embedded (FFPE) cell(s), acetone fixed cell(s), a paraformaldehyde (PFA) fixed cell(s), and / or a methanol fixed cell(s). The method can comprise: permeabilizing the sample and / or fixing the sample. Fixing the sample can comprise contacting the sample with a fixing agent. The fixing agent can comprise a non-cross-linking fixative (e.g., methanol). The fixing agent can comprise a cross-linking agent. The cross-linking agent can comprise a cleavable cross-linking agent. The cleavable cross-linking agent can comprise or can be derived from dithiobis(succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST). Bis [2-(Succinimidooxycarbonyloxy)ethyl] Sulfone (BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), dimethyl 3.3'- dithiobispropionimidate (DTBP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate (LC-SPDP), 4- succinimidyloxycarbonyl-alpha-methyl-a(2-pyridyldithio)toluene (SMPT), 3-(2- pyridyldithio)propionyl hydrazide (PDPH), succinimidyl 2-((4,4'-azipentanamido)ethyl)-1.3'- dithiopropionate (SDAD, NHS-SS-Diazirine), or any combination thereof. The cleavable crosslinking agent can comprise a cleavable linkage selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, an acid labile linker, a heat sensitive linkage, an enzymatically cleavable linkage, and a combination thereof. The cleavable cross-linking agent can be a thiol-cleavable cross-linking agent or can comprise a disulfide linker. The fixing agent can comprise paraformaldehyde (PF A), dithiobis(succinimidyl propionate (DSP), succinimidyl 3-(2-pyridyldithio)propionate (SPDP), CellCover, or a combination thereof. Fixing the sample and permeabilizing the sample can be carried out simultaneously.

[0183] Fixing and permeabilizing the sample can be carried out in the presence of a dual function agent capable of fixing and permeabilizing the sample. The dual functional agent can be methanol. Permeabilizing the sample can comprise contacting the sample with a permeabilizing agent. The method can comprise: after contacting the plurality of probing oligonucleotides or the plurality of cellular component-binding reagents with the sample, removing the permeabilizing agent from the sample. The permeabilizing agent can be capable of (i) permeabilizing the cell membrane of the cell(s), (ii) making a cell membrane of the cell(s) permeable to the probing oligonucleotides or the cellular component-binding reagents, or both. The permeabilizing agent can comprise (i) a solvent, a detergent, or a surfactant; (ii) BD Cytoperm; (iii) a saponin or a derivative thereof; (iv) Triton X-100, (v) methanol or a derivative thereof, and / or (vi) digitonin or a derivative thereof. The agent capable of dissociating protein- nucleic acid complexes can comprise a broad-spectrum serine protease. The broad-spectrum serine protease can be proteinase K. The lysis buffer can comprise an unfixing agent. The unfixing agent can comprise a thiol, hydoxylamine, periodate, a base, or any combination thereof. The lysis buffer can comprise DTT. The method can comprise: reversing the fixation of the sample and / or single cells. Reversing the fixation of the sample and / or single cells can comprise UV photocleaving, chemical treatment, heating, enzyme treatment, or any combination thereof.Blocking, Reagents. Decoy Oligonucleotides, and Blocking Oligonucleotides

[0184] The method can comprise: prior to contacting a plurality of cellular component-binding reagents with the sample and / or contacting the plurality of probing oligonucleotides with the sample, contacting the sample with a blocking reagent, one or more decoy oligonucleotides, and / or one or more blocking oligonucleotides. The methods and compositions provided herein can be employed in concert with the methods and compositions described in PCT Patent Application Number PCT / US22 / 75661, filed on August 30, 2022, entitled "RNA PRESERVATION AND RECOVERY FROM FIXED CELLS”, the content of which is incorporated herein by reference in its entirety. The methods and compositions provided herein can be employed in concert with blocking reagents, such as those described in PCT Patent Application Number PCT / US22 / 75656, filed on August 30, 2022, entitled “USE OF DECOY POLYNUCLEOTIDES IN SINGLE CELL MULTIOMICS”, the content of which is incorporated herein by reference in its entirety. Contacting a plurality of cellular component-binding reagents with the sample can be conducted in the presence of a blocking reagent. The blocking reagent can comprise a plurality of oligonucleotides complementary to at least a portion of the cellular component-binding reagent specific oligonucleotides. The blocking reagent can comprise an antibody or a fragment thereof derived from a first species, and the blocking reagent can comprise sera derived from the first species. The sample can comprise one or more non-target nucleic acids, and the blocking reagent can comprise a plurality' of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acids. Each of the plurality of decoy oligonucleotides can be capable of hybridizing to at least a portion of a non-target nucleic acid. In some embodiments, the decoy oligonucleotide: comprises a sequence complementary to at least a portion of a non-target nucleic acid; comprises a sequence identical to or substantially similar to a sequence of the cellular component-binding reagents specific oligonucleotides, optionally the sequence is 3-40 nucleotides in length; has at most 50% sequence identity to the cellular component-binding reagent specific oligonucleotides; does not comprise a UMI; comprises a random sequence, and optionally the random sequence is about four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen nucleotides in length; does not comprise any sequence having more than four, five, six, or seven consecutive Ts or As; comprise at least one G or C in every' four, five, six. or seven consecutive nucleotides; comprises one or more modified nucleotides; comprises a 5’ modification, and optionally the 5’ modification comprises a 5’ Amino Modifier C12 modification (5AmMC12); comprises a 3’ modification, and optionally the 3’ modification comprises a 3' dideoxy-C modification (ddC); and / or is 30 to 65 nucleotides in length.

[0185] The sample can comprise one or more undesirable nucleic acid species, and the method can comprise: contacting a blocking oligonucleotide with the sample, wherein the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleic acid species, and whereby the reverse transcription of the at least one of the one or more undesirable nucleic acid species is reduced by the blocking oligonucleotide. In some embodiments, the blocking oligonucleotide: is contacted with the sample before the plurality of probing oligonucleotides is contacted with the sample; is contacted with the sample after the plurality of probing oligonucleotides is contacted with the sample; and / or is contacted with the sample when the plurality of probing oligonucleotides is contacted with the sample. The method can comprise: providing blocking oligonucleotides that specifically bind to two or more undesirable nucleic acid species, optionally at least 10 or to at least 100 undesirable nucleic acid species, in the sample. In some embodiments, the blocking oligonucleotide: is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera; specifically binds to within 100 nt, to within 50 nt, or towi thin 25 nt of the 3’ end of the one or more undesirable nucleic acid species; specifically binds to within 100 nt of the 5’ end of the one or more undesirable nucleic acid species, or the blocking oligonucleotide specifically binds to within 100 nt of the middle of the one or more undesirable nucleic acid species; comprises or does not comprise non-natural nucleotides; has a Tm of at least 50 °C. of at least 60 °C. or of at least 70 °C; is unable to function as a primer for a reverse transcriptase or a polymerase; and / or is 8 nt to 100 nt long, is 10 nt to 50 nt long, is 12 nt to 21 nt long, is 20 nt to 30 nt long, or is about 25 nt long. The one or more undesirable nucleic acid species can amount to about 50%, to about 60%, to about 70%, or to about 80% of the nucleic acid content of the sample. The undesirable nucleic acid species can be selected from the group consisting of ribosomal RNA, mitochondrial RNA, genomic DNA, intronic sequence, high abundance sequence, and a combination thereof. The one or more undesirable nucleic acid species can be mRNA molecules and the blocking oligonucleotide can specifically binds to within 10 nt of the 3' poly (A) tail of the one or more undesirable nucleic acid species.Kits

[0186] There are provided, in some embodiments, compositions (e.g., kits). In some embodiments, the kit comprises: a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize a nucleic acid target, optionally the probing oligonucleotides comprise a predetermined spatial label; a coupling oligonucleotide comprising a 5’ complement of the coupling sequence and a 3’ complement of a capture sequence; a plurality of oligonucleotide barcodes, wherein the 3‘ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes is associated with a solid support, wherein the 5’ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a capture sequence; a first primer capable of hybridizing to the first universal sequence, optionally further comprising a third universal sequence; amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, optionally further comprising a second universal sequence; a DNA ligase; extension reagents, optionally reverse transcription reagents, further optionally a reverse transcriptase and dNTPs; one or more fixing agents; one or more permeabilizing agents; a crosslinking agent; unfixing agent; lysis buffer; a plurality of cellular component-binding reagents, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is capable of specifically binding to at least one of a plurality of cellular component targets; a blocking reagent; one or more decoy oligonucleotides; and / or one or more blocking oligonucleotides.

[0187] The plurality of probing oligonucleotides can comprise a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality7comprises a probe sequence configured hybridize a nucleic acid target of a plurality of nucleic acid targets, optionally a target panel of at least about 2, about 3. about 4, about 5. about 6, about 7. about 8, about 9, about 10, about 12. about 14. about 16, about 18, about 20. about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, or a number or a range between any two of these values, distinct nucleic acid targets.

[0188] The amplification primer(s) can comprise a panel of amplification primers configured to hybridize a plurality7of nucleic acid targets, or complements thereof, optionally a panel of at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12. about 14, about 16, about 18, about 20. about 30, about 40, about 50, about 60. about 70, about 80, about 90, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475, about 500, or a number or a range between any two of these values, distinct amplification primers.Terminology

[0189] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0190] One skilled in the art will appreciate that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods can be implemented in differing order. Furthermore, the outlined steps and operations are only provided as examples, and some of the steps and operations can be optional, combined into fewer steps and operations, or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments.

[0191] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The varioussingular / plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms ‘'a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0192] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory' phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g, the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g, “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone. B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A. B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms,or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0193] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0194] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken dow n into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3. 4, or 5 articles, and so forth.

[0195] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for labeling nucleic acid targets in a sample, comprising: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target, extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target; and barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target.

2. The method of claim 1, further comprising: obtaining sequencing data comprising a plurality' of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target; and determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.

3. A method for determining the copy number of a nucleic acid target in a sample, comprising: contacting a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize the nucleic acid target, extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target; barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, anda sequence complementary to at least a portion of the nucleic acid target; obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and a subsequence of the nucleic acid target; and determining the copy number of the nucleic acid target in the sample based on the number of molecular labels associated with the plurality of barcoded probing oligonucleotides, or products thereof.

4. A method for determining the spatial location and copy number of a nucleic acid target in a sample, comprising: contacting each of two or more spatial locations of a sample comprising copies of a nucleic acid target with a plurality of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence, a probe sequence configured hybridize the nucleic acid target, and a predetermined spatial label, wherein probing oligonucleotides contacted with the same spatial location comprise the same spatial label sequence, and wherein probing oligonucleotides contacted with distinct spatial locations of the sample comprise different spatial label sequences; extending the plurality of probing oligonucleotides hybridized to the copies of a nucleic acid target to generate a plurality of extended probing oligonucleotides each comprising a sequence complementary to at least a portion of the nucleic acid target; barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes to generate a plurality of barcoded probing oligonucleotides, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a molecular label, and wherein each of the plurality of barcoded probing oligonucleotides comprise a molecular label, a probe sequence, and a sequence complementary to at least a portion of the nucleic acid target; obtaining sequencing data comprising a plurality of sequencing reads of the barcoded probing oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a spatial label sequence, a molecular label sequence, and a subsequence of the nucleic acid target; and for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with a nucleic acid target to determine the copy number of the nucleic acid target at each spatial location of the sample.

5. The method of any one of claims 1-4, wherein barcoding the plurality of extended probing oligonucleotides, or products thereof, using a plurality of oligonucleotide barcodes comprises: providing a coupling oligonucleotide comprising a 5’ complement of the coupling sequence and a 3’ complement of a capture sequence; hybridizing the coupling sequence of the extended probing oligonucleotide with the 5 ’ complement of the coupling sequence of the coupling oligonucleotide; hybridizing the 3’ complement of the capture sequence of the coupling oligonucleotide with a capture sequence of an oligonucleotide barcode of the plurality of oligonucleotide barcodes; and ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode.

6. The method of any one of claims 1-5, comprising, before ligating the extended probing oligonucleotide to the oligonucleotide barcode, filling a gap between the extended probing oligonucleotide and the hybridized oligonucleotide barcode with a DNA polymerase lacking at least one of 5’ to 3’ exonuclease activity and 3’ to 5’ exonuclease activity7.

7. The method of any one of claims 1-6, wherein ligating the extended probing oligonucleotide to said hybridized oligonucleotide barcode is performed with a DNA ligase.

8. The method of any one of claims 1-7, wherein the coupling oligonucleotide is a single-stranded oligonucleotide, optionally the coupling oligonucleotide comprises at least 6 nucleotides.

9. The method of any one of claims 1-8. wherein the coupling sequence comprises at least 4 nucleotides.

10. The method of any one of claims 1-9, wherein the 5’ end of each probing oligonucleotide is phosphorylated.

11. The method of any one of claims 1-10, wherein the probing oligonucleotides are capable of entering a cell and / or a nucleus of the sample, optionally a permeabilized cell and / or a permeabilized nucleus of the sample.

12. The method of any one of claims 1-11, comprising after contacting the probing oligonucleotides with the sample, removing one or more probing oligonucleotides of the plurality of probing oligonucleotides that are not contacted with the sample, optionally removing the one or more probing oligonucleotides not contacted with the sample comprises: removing the one or more probing oligonucleotides that have not entered a cell of the sample.

13. The method of any one of claims 1-12, wherein the contacting step comprises contacting the sample with a device configured to deposit probing oligonucleotides, optionallyan inkjet device.

14. The method of any one of claims 1-13, wherein the device is a needle, a needle array, a tube, a suction device, an injection device, an electroporation device, a fluorescent activated cell sorter device, an inkjet device, a microfluidic device, or any combination thereof.

15. The method of any one of claims 1-14. wherein the device contacts distinct spatial locations of the sample at a specified rate.

16. The method of any one of claims 1-15, wherein the spatial label is 6-60 nucleotides in length.

17. The method of any one of claims 1-16. wherein said two or more spatial locations comprise at least about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, distinct spatial locations of the sample.

18. The method of any one of claims 1-17, wherein a spatial location of the sample corresponds to an area comprising no more than about 50 cells.

19. The method of any one of claims 1-18, comprising contacting the sample with extension reagents.

20. The method of any one of claims 1-19, wherein at least a portion of the contacting step is performed in the presence in the presence of extension reagents, optionally the entire contacting step is performed in the presence in the presence of the extension reagents, further optionally the contacting and extension steps are simultaneous.

21. The method of any one of claims 1-20, wherein the extension is performed in situ, optionally said extension comprises in situ reverse transcription, further optionally cells of the sample remain intact during the extension step.

22. The method of any one of claims 1-21, wherein the extension reagents comprise reverse transcription reagents, optionally reverse transcription reagents comprise a reverse transcriptase and dNTPs.

23. The method of any one of claims 1-22, wherein the reverse transcriptase comprises a viral reverse transcriptase, optionally wherein the viral reverse transcriptase is a murine leukemia virus (MLV) reverse transcriptase or a Moloney murine leukemia virus (MMLV) reverse transcriptase.

24. The method of any one of claims 1-23, wherein the sample is physically divided or is intact during the contacting step.

25. The method of any one of claims 1-24, wherein the sample comprises a single cell.

26. The method of any one of claims 1-25, wherein the sample comprises a pluralityof single cells.

27. The method of any one of claims 1-26, wherein the sample comprises a plurality of cells, and optionally the method comprises: disassociating the sample to generate a plurality of single cells, optionally said disassociating comprises chemical dissociating, enzymatic dissociating, and / or mechanical dissociating, optionally said disassociating employs one or more of collagenase, chymotrypsin, dispase, elastase, hyaluronidase, pancreatin, papain, and trypsin.

28. The method of any one of claims 1-27. comprising, prior to the barcoding step: partitioning the plurality7of single cells to a plurality of partitions, wherein a partition of the plurality of partitions comprises a single cell from the plurality of single cells; and in the partition comprising the single cell, contacting the extended probing oligonucleotide with the plurality of oligonucleotide barcodes.

29. The method of any one of claims 1-28, wherein in the partition comprising the single cell, contacting the single cell with a lysis buffer at 15-65 °C to lyse the single cell, optionally the lysis buffer comprises an agent capable of dissociating protein-nucleic acid complexes.

30. The method of any one of claims 1-29, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a first universal sequence.

31. The method of any one of claims 1-30, wherein obtaining sequencing data comprises: amplifying the plurality of barcoded probing oligonucleotides using a first primer capable of hybridizing to the first universal sequence or complement thereof, and amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, thereby generating a plurality of amplified barcoded probing oligonucleotides, wherein obtaining sequencing data comprises obtaining sequencing data comprising a plurality of sequencing reads of the amplified barcoded probing oligonucleotides, or products thereof.

32. The method of any one of claims 1-31, wherein obtaining sequencing data comprises attaching the binding sites of sequencing primers and / or sequencing adaptors to the plurality of barcoded probing oligonucleotides, or products thereof.

33. The method of any one of claims 1-32, wherein the amplification primer(s) comprise a second universal sequence and / or wherein the first primer comprises a third universal sequence.

34. The method of any one of claims 1-33, wherein:(i) the first universal sequence, the second universal sequence, and / or the third universal sequence are the same; and / or(ii) the first universal sequence, the second universal sequence, and / or the third universal sequence are different.

35. The method of any one of claims 1-34, wherein the first universal sequence, the second universal sequence, and / or the third universal sequence comprise the binding sites of sequencing primers and / or sequencing adaptors, complementary sequences thereof, and / or portions thereof.

36. The method of any one of claims 1-35, wherein the sequencing adaptors comprise a P5 sequence, a P7 sequence, complementary sequences thereof, and / or portions thereof.

37. The method of any one of claims 1-36, wherein the sequencing primers comprise a Read 1 sequencing primer, a Read 2 sequencing primer, complementary sequences thereof, and / or portions thereof.

38. The method of any one of claims 1-37, wherein the sample comprises a plurality of nucleic acid targets, optionally a target panel of at least about 2 distinct nucleic acid targets.

39. The method of any one of claims 1-38, wherein two or more nucleic acid targets of the target panel are biomarkers, optionally the biomarkers are biomarkers for a disease or condition, further optionally the disease or condition is a cancer, an infection, a viral infection, an inflammatory disease, a neurodegenerative disease, a fungal disease, a bacterial infection, or any combination thereof.

40. The method of any one of claims 1-39, wherein the contacting step comprises contacting the sample with a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality comprises a probe sequence configured hybridize a nucleic acid target of the plurality of nucleic acid targets.

41. The method of any one of claims 1-40, wherein determining the copy number of the nucleic acid target in the sample comprises determining the copy number of each of the plurality of nucleic acid targets in the sample based on the number of molecular labels with distinct sequences associated with the plurality of barcoded probing oligonucleotides, or products thereof, comprising a sequence of the each of the plurality of nucleic acid targets.

42. The method of any one of claims 1-41, comprising, for each unique spatial label sequence, which is associated with a distinct spatial location of the sample, counting the number of molecular labels with distinct sequences associated with each of the plurality7of nucleic acid targets to determine the copy number of each of the plurality of nucleic acid targets at each spatial location of the sample.-SO-43. The method of any one of claims 1-42, wherein the amplification primer(s) comprise a panel of amplification primers configured to hybridize the plurality of nucleic acid targets, or complements thereof, optionally a panel of at least about 2 distinct amplification primers.

44. The method of any one of claims 1-43, wherein the nucleic acid target comprises a nucleic acid molecule, optionally wherein the nucleic acid molecule comprises ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation product, RNA comprising a poly(A) tail, a sample indexing oligonucleotide, a cellular component-binding reagent specific oligonucleotide, or any combination thereof.

45. The method of any one of claims 1-44, wherein the plurality of cells comprise one or more cell types, optionally said one or more cell types are selected from the group consisting of: brain cells, heart cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, and circulatory cells, or any combination thereof.

46. The method of any one of claims 1-45, wherein the sample comprises a biological sample, a clinical sample, an environmental sample, a biological fluid, a tissue, a tissue section derived from a subject, or any combination thereof, optionally the subject is a human, a mouse, a dog, a rat, or a vertebrate.

47. The method of any one of claims 1-46, further comprising determining genotype, phenotype, or one or more genetic mutations of the subject based on the spatial location of the nucleic acid targets in the sample.

48. The method of any one of claims 1-47, further comprising predicting susceptibility of the subject to one or more diseases, optionally a cancer or a hereditary disease.

49. The method of any one of claims 1-48, further comprising determining cell types of the plurality of cells in the sample.

50. The method of any one of claims 1-49, wherein a drug is chosen based on predicted responsiveness of the cell types of the plurality of cells in the sample.

51. The method of any one of claims 1-50, comprising imaging the sample, optionally imaging the sample before the contacting step and / or after the contacting step, optionally the imaging generates imaging data.

52. The method of any one of claims 1-51. wherein imaging the sample comprises staining the sample with a stain, wherein the stain is a fluorescent stain, a negative stain, an antibody stain, or any combination thereof, optionally staining comprises Immunocytochemistry (ICC), Immunohistochemistry (IHC), Immunofluorescence (IF), or any combination thereof.

53. The method of any one of claims 1-52, wherein imaging comprises microscopy,confocal microscopy, time-lapse imaging microscopy, fluorescence microscopy, multi-photon microscopy, quantitative phase microscopy, surface enhanced Raman spectroscopy, videography, manual visual analysis, automated visual analysis, or any combination thereof.

54. The method of any one of claims 1-53, further comprising associating the imaging data and sequencing data of one or more spatial locations of the sample.

55. The method of any one of claims 1-54, comprising correlation analysis of the imaging data and the sequencing data of the spatial locations, optionally the correlation analysis identifies one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of therapeutic agents, and / or cellular targets of candidate therapeutic agents.

56. The method of any one of claims 1-55, wherein said imaging produces an image that is used to construct a map of a physical representation of said sample, optionally said map is two dimensional or three dimensional.

57. The method of any one of claims 1-56, comprising mapping the nucleic acid targets and / or cellular component targets onto the map of the sample.

58. The method of any one of claims 1-57, comprising mapping one or more single cells of the plurality of cells onto the map of the sample.

59. The method of any one of claims 1-58. wherein the sample has been contacted with one or more fixing agents and / or permeabilizing agents.

60. The method of any one of claims 1-59, wherein the sample comprises a tissue, a cell monolayer, fixed cells, a tissue section, or any combination thereof, optionally a fresh tissue section, a frozen tissue section, a fixed tissue section, a formalin-fixed tissue section, a formalin- fixed paraffin-embedded (FFPE) tissue section, an acetone fixed tissue section, a paraformaldehyde (PFA) fixed tissue section, and / or a methanol fixed tissue section.

61. The method of any one of claims 1-60, wherein the sample comprises a nuclei suspension, optionally a fixed nuclei suspension and / or a permeabilized nuclei suspension.

62. The method of any one of claims 1-61, wherein the sample comprises cell(s), optionally fresh cell(s), frozen cell(s), fixed cell(s), formalin-fixed cell(s). formalin-fixed paraffin-embedded (FFPE) cell(s), acetone fixed cell(s), a paraformaldehyde (PFA) fixed cell(s), and / or a methanol fixed cell(s).

63. The method of any one of claims 1-62, comprising: permeabilizing the sample and / or fixing the sample.

64. The method of any one of claims 1-63, wherein fixing the sample comprises contacting the sample with a fixing agent; optionally wherein the fixing agent comprises a noncross-linking fixative, and further optionally the non-cross-linking fixative comprises methanol; or optionally wherein the fixing agent comprises a cross-linking agent.

65. The method of claim 64, wherein the cross-linking agent comprises a cleavable cross-linking agent, and optionally wherein (a) the cleavable cross-linking agent comprises or is derived from dithiobis(succinimidyl propionate) (DSP), disuccinimidyl tartrate (DST), Bis [2- (Succinimidooxycarbonyloxy)ethyl] Sulfone (BSOCOES), ethylene glycol bis(succinimidyl succinate) (EGS), dimethyl 3.3'-dithiobispropionimidate (DTBP), succinimidyl 3-(2- pyridyldithio)propionate (SPDP), succinimidyl 6-(3(2-pyridyldithio)propionamido)hexanoate (LC-SPDP), 4-succinimidyloxycarbonyl-alpha-methyl-a(2-pyridyldithio)toluene (SMPT), 3-(2- pyridyldithio)propionyl hydrazide (PDPH), succinimidyl 2-((4,4'-azipentanamido)ethyl)-l,3'- dithiopropionate (SDAD, NHS-SS-Diazirine), or any combination thereof; (b) the cleavable cross-linking agent comprises a cleavable linkage selected from the group consisting of a chemically cleavable linkage, a photocleavable linkage, an acid labile linker, a heat sensitive linkage, an enzymatically cleavable linkage, and a combination thereof; and / or (c) the cleavable cross-linking agent is a thiol-cleavable cross-linking agent or comprises a disulfide linker.

66. The method of any one of claims 1-65, wherein the fixing agent comprises paraformaldehyde (PFA), dithiobis(succinimidyl propionate (DSP), succinimidyl 3-(2- pyridyldithio)propionate (SPDP), CellCover, or a combination thereof.

67. The method of any one of claims 1-66, wherein fixing the sample and permeabilizing the sample are carried out simultaneously.

68. The method of any one of claims 1-67, wherein fixing and permeabilizing the sample is carried out in the presence of a dual function agent capable of fixing and permeabilizing the sample, optionally wherein the dual functional agent is methanol.

69. The method of any one of claims 1-68, wherein permeabilizing the sample comprises contacting the sample with a permeabilizing agent.

70. The method of any one of claims 1-69, comprising, after contacting the plurality of probing oligonucleotides or the plurality7of cellular component-binding reagents with the sample, removing the permeabilizing agent from the sample.

71. The method of any one of claims 1-70. wherein: the permeabilizing agent is capable of (i) permeabilizing the cell membrane of the cell(s), (ii) making a cell membrane of the cell(s) permeable to the probing oligonucleotides or the cellular component-binding reagents, or both; the permeabilizing agent comprises (i) a solvent, a detergent, or a surfactant; (ii) BD Cytoperm; (iii) a saponin or a derivative thereof; (iv) Triton X-100, (v) methanol or a derivative thereof, and / or (vi) digitonin or a derivative thereof; the agent capable of dissociating protein-nucleic acid complexes comprises a broad-spectrum serine protease, optionally wherein the broad-spectrum serine protease isproteinase K; and / or the lysis buffer comprises (a) an unfixing agent, optionally wherein the unfixing agent comprises a thiol, hydoxylamine, periodate, a base, or any combination thereof; and / or (b) DTT.

72. The method of any one of claims 1-71. comprising reversing the fixation of the sample and / or single cells, optionally wherein reversing the fixation of the sample and / or single cells comprises UV photocleaving, chemical treatment, heating, enzyme treatment, or any combination thereof.

73. The method of any one of claims 1-72, wherein the sample comprises a plurality of cellular component targets, and wherein the method further comprises: contacting a plurality of cellular component-binding reagents with the sample, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is capable of specifically binding to at least one of the plurality of cellular component targets; barcoding the cellular component-binding reagent specific oligonucleotides to generate a plurality of barcoded cellular component-binding reagent specific oligonucleotides each comprising a sequence complementary to at least a portion of the unique identifier sequence and a molecular label sequence; and obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded cellular component-binding reagent specific oligonucleotides, or products thereof, wherein each of the plurality of sequencing reads comprises a molecular label sequence and at least a portion of the unique identifier sequence.

74. The method of any one of claims 1-73, wherein obtaining sequencing data comprises attaching the binding sites of sequencing primers and / or sequencing adaptors to the barcoded cellular component-binding reagent specific oligonucleotides, or products thereof.

75. The method of any one of claims 1-74, comprising after contacting the plurality of cellular component-binding reagents with the sample, removing one or more cellular component-binding reagents of the plurality of cellular component-binding reagents that are not contacted with the sample, optionally removing the one or more cellular component-binding reagents not contacted with the sample comprises: removing the one or more cellular component-binding reagents not contacted with the respective at least one of the plurality of cellular component targets.

76. The method of any one of claims 1-75, wherein the cellular component targetcomprises an intracellular protein, a carbohydrate, a lipid, a protein, an extracellular protein, a cell-surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

77. The method of any one of claims 1-76, wherein the cellular component-binding reagent specific oligonucleotide comprises a second molecular label, optionally at least ten of the plurality of cellular component-binding reagent specific oligonucleotides comprise different second molecular label sequences.

78. The method of any one of claims 1-77, wherein: the second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides are different, and wherein the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides are identical; and / or the second molecular label sequences of at least two cellular component-binding reagent specific oligonucleotides are different, and wherein the unique identifier sequences of the at least two cellular component-binding reagent specific oligonucleotides are different.

79. The method of any one of claims 1-78. wherein: the number of unique molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample; and / or the number of unique second molecular label sequences associated with the unique identifier sequence for the cellular component-binding reagent capable of specifically binding to the at least one cellular component target in the sequencing data indicates the number of copies of the at least one cellular component target in the sample.

80. The method of any one of claims 1-79, comprising, prior to contacting a plurality of cellular component-binding reagents with the sample and / or contacting the plurality of probing oligonucleotides with the sample, contacting the sample with a blocking reagent, one or more decoy oligonucleotides, and / or one or more blocking oligonucleotides.

81. The method of any one of claims 1-80, wherein contacting a plurality of cellular component-binding reagents with the sample is conducted in the presence of a blocking reagent.

82. The method of any one of claims 1-81, wherein the blocking reagent comprises a plurality7of oligonucleotides complementary to at least a portion of the cellular componentbinding reagent specific oligonucleotides.

83. The method of any one of claims 1-82, wherein the blocking reagent comprises an antibody or a fragment thereof derived from a first species, and wherein the blocking reagent comprises sera derived from the first species.

84. The method of any one of claims 1-83, wherein the sample comprises one or more non-target nucleic acids, wherein the blocking reagent comprises a plurality of decoy oligonucleotides capable of hybridizing to at least one of the one or more non-target nucleic acids.

85. The method of any one of claims 1-84, wherein each of the plurality of decoy oligonucleotides are capable of hybridizing to at least a portion of a non-target nucleic acid.

86. The method of any one of claims 1-85, wherein the decoy oligonucleotide: comprises a sequence complementary to at least a portion of a non-target nucleic acid; comprises a sequence identical to or substantially similar to a sequence of the cellular component-binding reagents specific oligonucleotides, optionally the sequence is 3-40 nucleotides in length; has at most 50% sequence identity' to the cellular component-binding reagent specific oligonucleotides; does not comprise a UMI; comprises a random sequence, and optionally the random sequence is about four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen nucleotides in length; does not comprise any sequence having more than four, five, six. or seven consecutive Ts or As; comprise at least one G or C in every four, five, six, or seven consecutive nucleotides; comprises one or more modified nucleotides; comprises a 5’ modification, and optionally the 5’ modification comprises a 5' Amino Modifier C12 modification (5AmMC12); comprises a 3’ modification, and optionally the 3’ modification comprises a 3' dideoxy-C modification (ddC); and / or is 30 to 65 nucleotides in length.

87. The method of any one of claims 1-86, wherein the sample comprises one or more undesirable nucleic acid species, the method comprising: contacting a blocking oligonucleotide with the sample, wherein the blocking oligonucleotide specifically binds to at least one of the one or more undesirable nucleicacid species; whereby the reverse transcription of the at least one of the one or more undesirable nucleic acid species is reduced by the blocking oligonucleotide.

88. The method of any one of claims 1-87. wherein the blocking oligonucleotide: is contacted with the sample before the plurality of probing oligonucleotides is contacted with the sample; is contacted with the sample after the plurality of probing oligonucleotides is contacted with the sample; and / or is contacted with the sample when the plurality of probing oligonucleotides is contacted with the sample.

89. The method of any one of claims 1-88, comprising providing blocking oligonucleotides that specifically bind to two or more undesirable nucleic acid species, optionally at least 10 or to at least 100 undesirable nucleic acid species, in the sample.

90. The method of any one of claims 1-89, wherein the blocking oligonucleotide: is a locked nucleic acid (LNA), a peptide nucleic acid (PNA), a DNA, an LNA / PNA chimera, an LNA / DNA chimera, or a PNA / DNA chimera; specifically binds to within 100 nt of the 3’ end of the one or more undesirable nucleic acid species; specifically binds to within 100 nt of the 5’ end of the one or more undesirable nucleic acid species, or the blocking oligonucleotide specifically binds to within 100 nt of the middle of the one or more undesirable nucleic acid species; comprises or does not comprise non-natural nucleotides; has a Tm of at least 50 °C; is unable to function as a primer for a reverse transcriptase or a polymerase; and / or is 8 nt to 100 nt long.

91. The method of any one of claims 1-90. wherein: the one or more undesirable nucleic acid species amounts to about 50% to about 80% of the nucleic acid content of the sample; the undesirable nucleic acid species is selected from the group consisting of ribosomal RNA, mitochondrial RNA, genomic DNA. intronic sequence, high abundance sequence, and a combination thereof; and / or the one or more undesirable nucleic acid species are mRNA molecules and the blocking oligonucleotide specific binds to within 10 nt of the 3‘ poly (A) tail of the one or more undesirable nucleic acid species.

92. The method of any one of claims 1-91, wherein: each molecular label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides; each capture sequence of the plurality of oligonucleotide barcodes comprises at least 4 nucleotides; the plurality of oligonucleotide barcodes are associated with a solid support, and wherein a partition of the plurality of partitions comprises a single solid support; the plurality of oligonucleotide barcodes each comprise a cell label, optionally each cell label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides; and / or oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with the same solid support comprise the same cell label, optionally oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with different solid supports comprise different cell labels.

93. The method of any one of claims 1-92, wherein the solid support comprises a synthetic particle, a planar surface, or a combination thereof.

94. The method of any one of claims 1-93. comprising associating a synthetic particle comprising the plurality of oligonucleotide barcodes with the cell in the partition.

95. The method of any one of claims 1-94, comprising lysing the cell after associating the synthetic particle with the cell, optionally lysing the cell comprises heating the cell, contacting the cell with a detergent, changing the pH of the cell, or any combination thereof; wherein the synthetic particle and the single cell are in the same partition, and optionally the partition is a well or a droplet; wherein at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is immobilized or partially immobilized on the synthetic particle, or at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is enclosed or partially enclosed in the synthetic particle; wherein the synthetic particle is disruptable, optionally a disruptable hydrogel particle; wherein the synthetic particle comprises a bead, optionally the bead comprises a Sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A / G conjugated bead, a protein L conjugated bead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorochrome microbead, orany combination thereof; and / or wherein the synthetic particle comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polysty rene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex. Sepharose, cellulose, nylon, silicone, and any combination thereof.

96. The method of any one of claims 1-95, wherein each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a linker functional group, wherein the synthetic particle comprises a solid support functional group, and wherein the support functional group and the linker functional group are associated with each other, and optionally the linker functional group and the support functional group are individually selected from the group consisting of C6, biotin, streptavidin, primary amine(s), aldehyde(s), ketone(s). and any combination thereof.

97. A kit comprising: a plurality7of probing oligonucleotides, wherein each of the probing oligonucleotides comprises a coupling sequence and a probe sequence configured hybridize a nucleic acid target, optionally the probing oligonucleotides comprise a predetermined spatial label; a coupling oligonucleotide comprising a 5’ complement of the coupling sequence and a 3’ complement of a capture sequence; a plurality of oligonucleotide barcodes, wherein the 3’ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes is associated with a solid support, wherein the 5’ end of each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprises a capture sequence; a first primer capable of hybridizing to the first universal sequence, optionally further comprising a third universal sequence; amplification primer(s) capable of hybridizing to the nucleic acid target or a complement a thereof, optionally further comprising a second universal sequence; a DNA ligase; extension reagents, optionally reverse transcription reagents, further optionally a reverse transcriptase and dNTPs; one or more fixing agents; one or more permeabilizing agents; a cross-linking agent; unfixing agent;lysis buffer; a plurality of cellular component-binding reagents, wherein each of the plurality of cellular component-binding reagents comprises a cellular component-binding reagent specific oligonucleotide comprising a unique identifier sequence for the cellular component-binding reagent, and wherein the cellular component-binding reagent is capable of specifically binding to at least one of a plurality of cellular component targets; a blocking reagent; one or more decoy oligonucleotides; and / or one or more blocking oligonucleotides.

98. The kit of claim 97, wherein the plurality of probing oligonucleotides comprises a panel of probing oligonucleotides comprising two or more pluralities of probing oligonucleotides wherein each plurality comprises a probe sequence configured hybridize a nucleic acid target of a plurality of nucleic acid targets, optionally a target panel of at least about 2 distinct nucleic acid targets.

99. The kit of any one of claims 97-98, wherein the amplification primer(s) comprise a panel of amplification primers configured to hybridize a plurality of nucleic acid targets, or complements thereof, optionally a panel of at least about 2 distinct amplification primers.

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