Methods and compositions for identifying tissue permeabilization conditions for producing spatial sequencing libraries

Abstract

The present disclosure is concerned with spatial detection of biological material, such as polynucleotides in a tissue sample or region thereof. The methods, compositions, and kits provided herein can be used to identify conditions useful for permeabilizing tissue to release biological material for subsequent interaction with immobilized capture molecules.

Description

IP-2912-PCT / 0531.002912WOO 1METHODS AND COMPOSITIONS FOR IDENTIFYING TISSUE PERMEABILIZATION CONDITIONS FOR PRODUCING SPATIAL SEQUENCING LIBRARIES

[0001] CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 739,169, filed December 27, 2024, the disclosure of which is incorporated by reference herein in its entirety.

[0003] FIELD

[0004] Embodiments of the present disclosure relate to preparing polynucleotides for spatial sequencing. In particular, embodiments of the methods, compositions, and kits provided herein relate to identifying conditions useful for permeabilizing tissue to release biological material, such as polynucleotides, for subsequent interaction with immobilized capture molecules.

[0005] BACKGROUND

[0006] Spatial transcriptomics enables highly multiplexed in situ gene expression profiling in which cellular relationships are captured within complex tissue architectures. To generate spatial sequencing libraries, an on-surface library preparation method should spatially capture and barcode transcripts from a tissue sample. One step of on-surface library preparation includes permeabilization, in which mRNA released and subsequently captured on the surface. Current spatial workflows, however, typically capture and convert <1 % mRNA within a tissue section.

[0007] SUMMARY OF THE APPLICATION

[0008] Provided herein are methods for identifying conditions useful for permeabilizing tissue. In one embodiment, a method includes providing a surface including a plurality of immobilized capture oligos, where the capture oligos are immobilized at the 5’ end and the capture oligos include a 3’ end region, and contacting the surface with a tissue including aIP-2912-PCT / 0531.002912WOO 1plurality of mRNA molecules in cells. The tissue is permeabilized to release mRNA molecules from the cells, where a region of the mRNA molecules anneals to the 3’ end region of the capture oligos. Reverse transcriptase (RT) is used to extend the 3’ end of the capture oligos using the annealed mRNA molecules as template to produce a first complementary sequence, where the RT adds non-templated nucleotides to the 3’ end of the first complementary sequence. A template switch oligo (TSO) can be annealed to the non-templated nucleotides. The 3’ end of the non-templated nucleotides can be extended using the 5’ end of the TSO as a template to result in a complementary TSO that is attached to the non-templated nucleotides and the first complementary sequence. The TSO can be annealed to the complementary TSO and then extended using the non-templated nucleotides and the first complementary sequence as a template to result in a second complementary sequence. At least a portion of the second complementary sequence can be amplified in the presence of a labeled dNTP to result in labeled polynucleotides immobilized to the surface, and the amount of the labeled dNTP associated with the surface can be determined.

[0009] In another embodiment, a method includes providing a surface including a plurality of immobilized capture oligos, where the capture oligos are immobilized at the 5’ end and the capture oligos include a 3’ end region, and contacting the surface with a tissue including a plurality of mRNA molecules in cells. The tissue is permeabilized to release mRNA molecules from the cells, where a region of the mRNA molecules anneals to the 3’ end region of the capture oligos. RT is used to extend the 3’ end of the capture oligos using the annealed mRNA molecules as template to produce a first complementary sequence, where the RT adds non-templated nucleotides to the 3’ end of the first complementary sequence. A primer is annealed to nucleotides of the first complementary sequence, and the primer 3’ end is extended using the first complementary sequence as a template to result in a second complementary sequence. At least a portion of the second complementary sequence can be amplified in the presence of a labeled dNTP to result in labeled polynucleotides immobilized to the surface, and the amount of the labeled dNTP associated with the surface can be determined.

[0010] BRIEF DESCRIPTION OF THE FIGURESIP-2912-PCT / 0531.002912WOO 1

[0011] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings.

[0012] FIG. 1 shows a general block diagram of a portion of a general illustrative workflow for determining permeabilization conditions when mRNA is being analyzed.

[0013] FIGs. 2-12 show schematic drawings of embodiments for identifying permeabilization conditions. For simplicity, only a limited area of a surface with immobilized capture oligos and other polynucleotides produced during practice of methods disclosed herein are shown.

[0014] FIG. 13 shows the results of flowcells grafted with a single capture oligo or co-grafted with two capture oligos.

[0015] FIG. 14 shows permeabilization results for mouse kidney tissue using ExAmp imaging.

[0016] FIG. 15 shows permeabilization results for mouse cerebellum tissue using ExAmp imaging.

[0017] FIG. 16 shows permeabilization results for mouse heart tissue using ExAmp imaging.

[0018] The schematic drawings are not necessarily to scale. Like numbers used in the figures refer to like components, steps and the like. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. In addition, the use of different numbers to refer to components is not intended to indicate that the different numbered components cannot be the same or similar to other numbered components.

[0019] DETAILED DESCRIPTION

[0020] The production of on-surface libraries requires permeabilization of tissue for release of molecules such as mRNA. Multiple factors, however, can affect permeabilization. Factors include, for instance, the type of tissue (e.g., small intestine vs. spleen), organism (e.g., human brain vs. mouse brain), developmental stage (e.g., embryonic vs. adult), disease state (e.g., healthy vs. diseased), tissue region (e.g., homogeneous vs. heterogeneous), section thickness, sectioning plane, and tissue quality. Suitable permeabilization conditionsIP-2912-PCT / 0531.002912WOO 1must also permit efficient capture of mRNA and subsequent conversion of captured mRNA into on-surface libraries. Only a small percentage of mRNA (<1 %) is typically captured and converted within a tissue section, thus identification of suitable permeabilization conditions can be helpful in increasing sensitivity. The present disclosure combines on- surface polynucleotide capture (e.g., mRNA), conversion to DNA, and amplification to convert captured tissue mRNAs into labelled amplicons, which can be assayed to identify suitable permeabilization conditions. Optionally, identified permeabilization conditions can then be used for the construction of on-surface libraries.

[0021] Provided herein are methods for determining permeabilization conditions. In some embodiments, the identified conditions can be used to produce on-surface libraries. A workflow suitable for determining permeabilization conditions can include (i) providing a surface with attached capture oligos, (ii) annealing polynucleotides (e.g., mRNA) to the capture oligos, (iii) using reverse transcriptase to extend the capture oligos using the annealed polynucleotides as templates to yield attached first complementary sequences, (iv) removing the annealed polynucleotides, (v) annealing a primer to non-templated nucleotides attached to the first complementary sequence, (vi) extending the non-templated nucleotides using the primer as template to yield an unattached second complementary sequence, (vii) amplifying the unattached second complementary sequence in presence of a labeled dNTP to yield attached amplicons, and (viii) determining amount of label associated with the amplicons (FIG. 1).

[0022] Surface

[0023] Methods of the present disclosure can include providing a surface with attached capture oligos (FIG. 1, block 10). As used herein, a “substrate” refers to any material that is appropriate for, or can be modified to be appropriate for, the attachment of the capture oligos. Examples of substrates include, but are not limit to, glass (including modified glass, functionalized glass), plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon or nitrocellulose, ceramics, resins, silica or silica- based materials including silicon and modified silicon, carbon, metals, inorganic glasses,IP-2912-PCT / 0531.002912WOO 1plastics, and a variety of other polymers. A substrate is generally rigid and is insoluble in an aqueous liquid.

[0024] A substrate can be in a form suitable for application of a tissue section. Examples of shapes include, but are not limited to, a microscope slide, a bead, a bead array, a spotted array, clustered particles arranged on a surface of a chip, a flow-cell, and a multi-well plate. A substrate can be non-pattemed, e.g., substantially planar. Alternatively, a surface can be patterned, e.g., include depressions.

[0025] Capture oligos

[0026] A surface includes a plurality of attached capture oligonucleotides (also referred to as capture oligos). As used herein, a “capture oligo” refers to a polynucleotide having a nucleotide sequence that can anneal to a single stranded polynucleotide sequence to be analyzed (e.g., an mRNA) and / or be subjected to a nucleic acid interrogation under conditions encountered in a primer annealing step of, for example, an amplification reaction. A capture oligo can be single stranded, double stranded, or have a mixture of one or more regions that are singled stranded and double stranded. In some embodiments a capture oligo is single stranded. A capture oligo can include natural nucleotides (adenine, cytosine, guanine, thymine, inosine, uracil), modified nucleotides (e.g., locked nucleic acids (LNAs), bridged nucleic acids (BNAs)), or any combination thereof. A capture oligo can include a nucleotide suitable for inducing cleavage of the capture oligo, and such nucleotides are described herein.

[0027] A capture oligo includes a 5' end that is attached (also referred to herein as immobilized) to the surface. The attachment allows the 3' end to be available for enzymatic extension and at least a portion of the sequence at the 3' to be available for hybridizing to a complementary sequence. The attachment is typically covalent.

[0028] The 5' end of each attached capture oligo includes a domain (5' domain) of nucleotide sequence that is substantially the same, and in one embodiment is identical in all capture oligos on a surface. The 5' domain does not need to be at the 5' end, and can be at least 1, at least 5, or at least 10 nucleotides from the 5' end of the capture oligo. This domain isIP-2912-PCT / 0531.002912WOO 1useful in the method to aid in amplification, for instance, amplification that includes strand invasion. Amplification is described herein.

[0029] A capture oligo can optionally include a 3' domain of nucleotide sequence that is designed to hybridize to mRNA. Examples of suitable nucleotides include, but are not limited to, a poly-T domain, a randomer domain, a target-specific domain, or a disrupted homopolymer. In some embodiments, a primer can include one or more inosine nucleotides. A poly-T domain is useful in hybridizing to the poly-A region of mRNAs. A randomer domain includes a random sequence of nucleotides of 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length that anneals to a random location on a mRNA.

[0030] As used herein, a “target-specific domain,” when used in reference to a capture oligo, refers to a 3’ domain of a capture oligo that includes a nucleotide sequence complementary to a targeted polynucleotide. For instance, nucleotides of a target-specific domain will selectively anneal to a targeted polynucleotide, e.g., a mRNA encoded by a specific coding region. A surface can include capture oligos with a target-specific domain, and optionally a surface can include different populations of capture oligos, each population having a single target-specific domain that targets a different set of nucleotides of the same mRNA.

[0031] As used herein, a "disrupted homopolymer" refers to a 3’ domain of a capture oligo that includes two or more nucleotides or two or more nucleotide sequences complementary to a mRNA poly-A region. In contrast to an uninterrupted series of consecutive thymidine nucleotides (e.g., a poly-T domain), a disrupted homopolymer includes at least two nonsequential nucleotides or non-sequential nucleotide sequences, where each of the nonsequential nucleotides or non-sequential nucleotide sequences is complementary to a portion of a mRNA poly-A region. Each of the non-sequential nucleotides or nonsequential nucleotide sequences are separated by an intervening nucleotide or intervening nucleotide sequence. For example, a disrupted homopolymer of a capture oligo can have a first series of three thymines, followed by a non-thymine nucleotide, and a second series of three thymines. Such patterns can be repeated multiple times within a 3' domain of a capture oligo. The presence of an intervening nucleotide or intervening nucleotide sequence between non-sequential nucleotides or non-sequential nucleotide sequences doesIP-2912-PCT / 0531.002912WOO 1not prevent hybridization between a mRNA poly-A region and complementary thymidine nucleotides present in a disrupted homopolymer.

[0032] A capture oligo can include a cleavage site. Any suitable enzymatic, chemical, or photochemical cleavage reaction can be used to cleave a capture oligo at a cleavage site. Cleavage can be achieved by, for example, nicking enzyme digestion, in which case the cleavage site is an appropriate restriction site for the enzyme which directs cleavage of the capture oligo; RNase digestion or chemical cleavage of a bond between a deoxyribonucleotide and a ribonucleotide, in which case the cleavage site can include one or more ribonucleotides; chemical reduction of a disulfide linkage with a reducing agent (e.g., TCEP), in which case the cleavage site should include an appropriate disulfide linkage; chemical cleavage of a diol linkage with periodate, in which case the cleavage site should include a diol linkage; and generation of an abasic site and subsequent hydrolysis.

[0033] Suitable cleavage techniques for use in the method of the disclosure include, but are not limited to, chemical cleavage, cleavage of an abasic site, cleavage of a ribonucleotide, photochemical cleavage, cleavage of hemimethylated DNA, PCR stoppers, cleavage of a peptide linker, and enzymatic digestion with nicking endonuclease.

[0034] Chemical cleavage encompasses any method which uses a non-nucleic acid and non- enzymatic chemical reagent to promote / achieve cleavage of a capture oligo. If required, a capture oligo may include one or more non-nucleotide chemical moieties and / or nonnatural nucleotides and / or non-natural backbone linkages in order to permit chemical cleavage reaction. In one embodiment, a capture oligo includes a diol linkage which permits cleavage by treatment with periodate (e.g., sodium periodate).

[0035] An abasic site is a position in a capture oligo from which the base component has been removed. Once formed, abasic sites can be cleaved (e.g., by treatment with an endonuclease or other single-stranded cleaving enzyme, exposure to heat or alkali), providing for site-specific cleavage the capture oligo.

[0036] In one embodiment, an abasic site can be created at a pre-determined position of the capture nucleic acid and then cleaved by first incorporating deoxyuridine (U) at the pre-IP-2912-PCT / 0531.002912WOO 1determined cleavage site. The enzyme uracil DNA glycosylase (UDG) can then be used to remove the uracil base, generating an abasic site. The strand including the abasic site may then be cleaved at the abasic site by treatment with endonuclease (e.g. EndoIV endonuclease, AP lyase, FPG glycosylase / AP lyase, EndoVIII glycosylase / AP lyase), heat or alkali.

[0037] Abasic sites may also be generated at non-natural / modified deoxyribonucleotides other than deoxyuridine and cleaved in an analogous manner by treatment with endonuclease, heat or alkali. For example, 8-oxo-guanine can be converted to an abasic site by exposure to FPG glycosylase. Deoxyinosine can be converted to an abasic site by exposure to AlkA glycosylase. The abasic sites generated may then be cleaved, typically by treatment with a suitable endonuclease (e g., EndoIV, AP lyase). In a particular embodiment, the USER (Uracil-Specific Excision Reagent) reagent available from New England Biolabs (NEB M55O5S) is used for the creation of a single nucleotide gap at a uracil base in a capture nucleic acid.

[0038] A capture oligo can be 8 to 80 nucleotides in length. In certain embodiments, the RNA capture probe or surface probe is 10 to 80 nucleotides, 10 to 70 nucleotides, 10 to 60 nucleotides, 10 to 50 nucleotides, 10 to 40 nucleotides, 10 to 30 nucleotides, 10 to 20 nucleotides, 20 to 80 nucleotides, 20 to 70 nucleotides, 20 to 60 nucleotides, 20 to 50 nucleotides, 20 to 40 nucleotides, or is 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nucleotides.

[0039] A surface can include one type of capture oligo, e.g., the surface includes a homogeneous lawn or field of the same capture oligo. A surface having a single population of capture oligo attached is referred to herein as a grafted surface. A surface can include more than type of capture oligo, e.g., the surface includes a heterogeneous lawn or field of two or more capture oligos. A surface having two or more populations of capture oligos attached is referred to herein as a co-grafted surface. Thus, a surface can include 1, 2, 3, 4, or 5 different capture oligos. In one embodiment, a surface includes 1 or 2 populations of capture oligos. Examples of grafted and co-grafted surfaces are shown in FIG. 2. FIG. 2A shows a grafted surface 20 having one population of capture oligos 21 attached by the 5'IP-2912-PCT / 0531.002912WOO 1end to the surface. Each capture oligo 21 includes a 5' domain 22 and a 3' domain 23. FIG. 2B shows a co-grafted surface 20 having two population of capture oligos, each attached by the 5' end to the surface. One population of capture oligo 21 includes a 5' domain 22 and a 3' domain 23, and a second population of capture oligo 24 includes a 5' domain 22. In one embodiment, the 5' domain 22 of each population of capture oligo 21 and 24 is the same.

[0040] A surface can have a capture site density of 1 or more, 2 or more, 10 or more, 30 or more,100 or more, 300 or more, 1,000 or more, 3,000 or more, 10,000 or more, 100,000 or more, 1,000,000 or more, capture sites per square centimeter (cm2).

[0041] Optionally, a capture oligo can include a barcode (e g., a spatial barcode), a unique molecular identifier (UMI), or both barcode and UMI. In one embodiment, a capture oligo does not include a barcode, does not include a UMI, or does not include both barcode and UMI.

[0042] Tissue

[0043] The method includes annealing mRNA molecules to the capture oligos present on a surface. The mRNA molecules are from a biological sample, such as a tissue.

[0044] A tissue may be sectioned, e.g., subjected to a process of cutting thin uniform slices of tissue, and individual sections used in a method of the present disclosure. Methods for sectioning tissue are known in the art. The thickness of a tissue sample or other biological sample that is contacted with a surface in a method set forth herein can be any suitable thickness desired. In some embodiments, the thickness is at least 0.1 pm, 0.25 pm, 0.5 pm, 0.75 pm, 1 pm, 5 pm, 10 pm, 50 pm, 100 pm or thicker. In some embodiments, the thickness of a biological sample that is contacted with a surface will be no more than 100 pm, 50 pm, 10 pm, 5 pm, 1 pm, 0.5 pm, 0.25 pm, 0.1 pm or thinner. In one embodiment, a tissue section can be contacted with a surface, for example, by laying the tissue on the surface. Optionally, a tissue section can be attached to a surface, for example, using techniques and compositions described in, for example, U.S. Patent No. 11,390,912.

[0045] PermeabilizingIP-2912-PCT / 0531.002912WOO 1

[0046] Methods of the present disclosure include permeabilizing the tissue present on a surface.As used herein, “permeabilizing” tissue refers to releasing molecules present in a tissue, e.g., intracellular and intercellular molecules, and subsequent capture of molecules, e.g., polynucleotides, by capture oligos on the surface. Examples of polynucleotides released from tissue include, but are not limited to, mRNA, gDNA, rRNA, tRNA, or a combination thereof.

[0047] Typically, conditions useful for permeabilizing one tissue do not work effectively with other tissues, thus the permeabilizing can include identifying conditions that release molecules from a tissue and permit capture of the molecules by the surface capture oligos. Identifying conditions for permeabilizing tissue can include, but are not limited to, varying the amount or concentration of a permeabilization agent, varying the time of the treatment, varying the temperature of the treatment, varying the thickness of the tissue, using different combinations of permeabilization agents, or a combination of thereof.

[0048] Examples of permeabilization agents include, but are not limited to, organic solvents, detergents, and enzymes. Examples of organic solvents include, but are not limited to, methanol, acetone, chloroform, and dichloromethane. Examples of detergents include, but are not limited to, NP40, streptolysin O, a saponin, digitonin, Triton™ X-100, Tween®-20, Leucoperm™, 3-[(3-cholamidopropyl)dimethylammonio]-l -propanesulfonate hydrate (CHAPS), or dodecyltrimethylammonium chloride (DOTMAC). Examples of enzymes include, but are not limited to, proteinase K or streptolysin O (SLO).

[0049] The concentration of the permeabilization agent can vary depending on the agent used. In general, organic solvents can be used at concentrations of at least 0.0001 moles / liter (M), at least 0.001 M, at least 0.01 M, or at least 0.1 M, to no greater than 2 M, no greater than 1 M, or no greater than 0.1 M. In general, detergents are typically in a buffer, and can be used concentrations of at least 0.01%, at least 0.1%, at least 1%, at least 2.5%, or at least 5%, to no greater than 7.5%, no greater than 5%, no greater than 2.5%.

[0050] The time of the treatment can vary from 1 minute to 60 minutes, and in some embodiments can be greater than 60 minutes. For instance, the treatment can occur for 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,IP-2912-PCT / 0531.002912WOO 133, 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, or 60 minutes.

[0051] The temperature of the treatment can vary from at least 4 °C, at least 10 °C, at least 20 °C, at least 30 °C, at least 40 °C, at least 49 °C, to no greater than 50 °C, no greater than 40 °C, no greater than 30 °C, no greater than 20 °C, to no greater than 10 °C.

[0052] Optionally, a tissue section can be stained using standard tissue staining reagents including, but not limited to, hematoxylin and eosin. Staining can be used for histological analysis of a tissue. The staining can be done before or after permeabilization.

[0053] In some embodiments, a polynucleotide such as mRNA anneals to nucleotides present on the 3' domain of a capture oligo. As described herein, examples of suitable nucleotides present on a capture oligo 3' domain include, but are not limited to, a poly-T domain, a randomer domain, a target-specific domain, or a disrupted homopolymer. An illustration of a mRNA annealing to a capture oligo is depicted in FIG. 3. mRNA 30 includes a poly-A tail 31 that anneals to a poly-T region of the 3' domain 23 of capture oligo 21.

[0054] Optionally, a tissue section is removed from the surface after permeabilization. In one embodiment, a tissue is removed by enzymatic degradation. Tissue sections can be removed by, for instance, degradation with proteinase K.

[0055] Reverse transcription

[0056] The method includes use of a reverse transcriptase to produce a first complementary sequence. Reverse transcriptase extends the 3’ end of the capture oligo using the annealed mRNA as a template.

[0057] The result of reverse transcription is a first complementary sequence that includes the capture oligo and a DNA sequence that is complementary (cDNA) to the annealed mRNA. The first complementary sequence attached to the surface. When the annealed molecule is mRNA and the capture oligo 3' domain includes a poly-T region or disrupted homopolymer, the first complementary sequence includes the complement of the full length of the mRNA. In other embodiments, for instance when the capture oligo 3' domainIP-2912-PCT / 0531.002912WOO 1includes a randomer domain or a target-specific domain, the first complementary sequence can include the complement of a portion the mRNA sequence.

[0058] An illustration of an extension 30' of a capture oligo by reverse transcriptase is depicted in FIG. 3. An example of the result of reverse transcription is depicted in FIG. 4. The 3' domain 23 of capture oligo 21 has been extended to result in a first complementary sequence 32. First complementary sequence 32 includes the complement of mRNA 30, the capture oligo 21, and is attached to surface 20.

[0059] In some embodiments, synthesis of the first complementary sequence includes a method for template switching. Template switching typically includes the use of a reverse transcriptase that adds nucleotides to the end of the 3’ end of the extended strand, and a template switching oligo (TSO) that acts as a primer for extension of a new strand that uses the first complementary sequence as template. Examples of reverse transcriptases that add nucleotides to the extended strand (e.g., the first complementary sequence) include, but are not limited to, Moloney Murine Leukemia Virus Reverse Transcriptase. The number of nucleotides added, also referred to as non-template nucleotides, can be 2, 3, or more. The nucleotides added are typically the same, e.g., 2 or more cytosines, 2 or more adenines, 2 or more guanines, or 2 or more thymidines. The added nucleotides at the 3' end of the extended strand produce a DNA / RNA hybrid with a single stranded DNA overhang at the 3' end of the DNA strand. An illustration of the result of reverse transcription with addition of non-templated nucleotides is depicted in FIG. 5. The 3' domain 23 of capture oligo 21 has been extended to result in a first complementary sequence 32 that also includes three non-templated cytidine nucleotides at the 3’ end.

[0060] The overhang can provide a nucleotide sequence to which complementary nucleotides can anneal and provide an additional template (e.g., a template switching oligo) for further extension of the first complementary sequence. In some embodiments, the complementary nucleotides that anneal to the first complementary sequence 32 overhang contains a template switching oligo, the complement of which is incorporated into the first complementary sequence. FIG. 6 depicts an embodiment where complementary guanines anneal to the non-template cytosines. The complementary guanines include an additionalIP-2912-PCT / 0531.002912WOO 1template 37. The reverse transcriptase uses the additional template 37 to further extend the first complementary sequence 32 to result in a nucleotide sequence 38 added to the 3’ end of the first complementary sequence 32.

[0061] The method can include an optional step of removing the annealed polynucleotide, e.g.RNA, after synthesis of the first complementary sequence. In those embodiments using a template switching oligo, removing the annealed polynucleotide can include removal of the template switching oligo. For example, a RNA digesting enzyme (RNase) e.g. RNase H can be used. Methods for using RNAse are known in the art. This is generally not needed, however, and in most cases the RNA degrades naturally. Removal of the tissue sample from the array will generally remove the RNA from the array. A RNase can used if desired to increase RNA removal. Removal of RNA may provide a consistent target to which a primer for second strand synthesis can bind, e.g., all of the immobilized molecules will be single stranded after RNA removal. Prior to a step of RNA removal, the immobilized molecules may be a mixture of fully or partially double stranded molecules (RNA-DNA hybrids) and single stranded molecules (where the RNA has already degraded). In those embodiments using a template switching oligo, removing the annealed polynucleotide can include removal of the template switching oligo.

[0062] Second strand synthesis

[0063] The method includes synthesis of a second complementary sequence (also referred to herein as “second strand synthesis”) that is complementary to the first complementary sequence. In some embodiments, a primer is added to a surface having a plurality of attached first complementary sequences. After annealing of the primer to the first complementary sequence a DNA polymerase can be used to extend the 3’ end of the primer using the first complementary sequence as a template. In some embodiments the polymerase has strand displacing activity. Examples of primers include, but are not limited to, a randomer primer, a target-specific primer, a primer comprising one or more inosines, or a template-switching oligo. Thus, in some embodiments second strand synthesis can be achieved by template switching. A randomer primer is similar to a randomer domain described herein; it includes a random sequence of nucleotides of 2, 3, 4, 5, 6, 7, 8, 9, or 10IP-2912-PCT / 0531.002912WOO 1nucleotides in length that anneals to a random location on a first complementary sequence. A target-specific primer is similar to a target-specific domain described herein; it includes a nucleotide sequence specific to a targeted polynucleotide. For instance, nucleotides of a target-specific primer will selectively anneal to a targeted polynucleotide, e.g., a mRNA encoded by a specific coding region. A single target-specific primer can be used, or different populations of target-specific primers can be used, where each population has a single target-specific domain that targets a different set of nucleotides of the same mRNA.

[0064] An illustration of second strand synthesis is depicted in FIG. 7. The surface 20 includes first complementary sequences that were produced using a template switching oligo. The first complementary sequence 32 includes the nucleotide sequence 38 at the 3’ end, where the nucleotide sequence is the complement of a template switching oligo (e.g., template switching oligo 37 of FIG. 6). The template switching oligo 37 is annealed to the 3’ end of first complementary sequence 32 and extended 33 using the first complementary sequence 32 as a template. The result of the strand synthesis is depicted in FIG. 8, where the extension is complete and yields a second complementary sequence 35 that is not attached to surface 20. Second complementary sequence 35 includes the template switching oligo 37, the DNA sequence of the mRNA (e.g., mRNA 30 of FIG.3), and the complement of the capture oligo 21.

[0065] Amplification

[0066] The method further includes amplification of the second complementary sequence. In some embodiments, amplification is by kinetic exclusion, where recombinase-facilitated amplification and isothermal conditions amplify the library (U.S. Pat. No. 9,309,502, U.S. Pat. No. 8,895,249, U.S. Pat. No. 8,071,308). Methods that rely on kinetic exclusion are referred to as kinetic exclusion amplification (KEA), exclusion amplification (ExAmp), or kinetic amplification. The amplification can include adding an amplification reagent to a surface that has a plurality of attached first complementary sequences and hybridized unattached second complementary sequences. An amplification reagent can include components that facilitate amplicon formation. Recombinase, example UvsX, can facilitate amplicon formation by allowing repeated invasion / extension. More specifically,IP-2912-PCT / 0531.002912WOO 1recombinase can facilitate invasion of a double stranded polynucleotide (e.g., an attached first complementary sequence and hybridized unattached second complementary sequence) by an attached capture oligo and extension of the capture oligo by a polymerase using the target nucleic acid as a template for amplicon formation. In one embedment, the polymerase is a strand-displacing polymerase. This process can be repeated as a chain reaction where amplicons produced from each round of invasion / extension serve as templates in a subsequent round. The process can occur more rapidly than standard PCR since a denaturation cycle (e.g., via heating or chemical denaturation) is not required. As such, recombinase-facilitated amplification can be carried out isothermally. A mixture of recombinase and single-stranded binding (SSB) protein is particularly useful as SSB can further facilitate amplification. Exemplary formulations for recombinase-facilitated amplification include those sold commercially as TwistAmp kits by TwistDx (Cambridge, UK). Useful components of recombinase-facilitated amplification reagent and reaction conditions are set forth in U.S. Pat. No. 5,223,414 and U.S. Pat. No. 7,399,590.

[0067] The amplification reagent can include, but is not limited to, nucleotide triphosphates (NTPs). The NTPs include dATP, dTTP, dCTP, dGTP, or a subset of these NTPs, e.g., just one dNTP, two of the dNTPs, or three dNTPs. In one embodiment, one or more of the dNTPs is labeled. "Label" refers to a detectable marker that is attached to one or more dNTP. Examples of labels include, but are not limited to, labels that can be identified imaging (e.g., by observation with a microscope) or by a device that allows quantification of the amount of label (e.g., a plate reader or a slide scanner). Examples of labels include, but are not limited to, fluorescent dyes, radioactive isotopes, enzyme tags, biotinylation, and antibody. The combination of amplification and incorporation of label during amplification permits boosting the signal and increasing the sensitivity of the method in identifying permeabilization conditions.

[0068] An illustration of recombinase-facilitated amplification is depicted in FIG. 9. The surface 20 includes attached first complementary sequence 32 that includes the nucleotide sequence 38 at the 3’ end and hybridized unattached second complementary sequences 35.A capture oligo, which includes 5' domain 22 and 3' domain 23, invades the double stranded structure of attached first complementary sequence and hybridized unattachedIP-2912-PCT / 0531.002912WOO 1second complementary sequence. The invading capture oligo strand hybridizes to the region of the unattached second complementary sequence 35 that is a complement of the invading strand, and the 3' end of the invading strand is extended 40 using the second complementary sequence 35 as a template. When the extension occurs in the presence of one or more labeled dNTPs, the extended sequence 40 incorporates the labeled dNTPs (depicted as stars in FIG. 9). The extension 40, which typically occurs by stranddisplacing polymerase, continues until the end of the template second complementary sequence 35. This process is repeated as a chain reaction where the amplicon produced from each round of invasion / extension serves as a double stranded structure of attached first complementary sequence 32 (now including labeled dNTPs) and hybridized unattached second complementary sequence 35 for another round of amplification. The result is shown in FIG. 10, where multiple attached and labeled first complementary sequences 32 have been produced.

[0069] Using a first complementary sequence as template for addition of labeled dNTPs as depicted in FIGs. 9-10 can result in different amounts of label depending on the ratios of the four different nucleotides in each different first complementary sequence present on a surface. Labeling only nucleotides complementary to a 3' domain of capture oligo reduces or completely removes this variation, and results in a more consistent amount of labeling across a surface. Accordingly, in some embodiments the capture oligos of a co-graft surface can be configured for the production of labeled extension products that use only a 3' domain of a capture oligo as template. For instance, a co-graft surface can be used, such as the co-graft surface depicted in FIG. 2B. The 3' domain 23 of a capture oligo 21 can be configured to include a specific dNTP near the 3' end, and the mixture of dNTPs used in the recombinase-facilitated amplification can exclude the complement of that specific dNTP. The result of extension after invasion is a truncated polynucleotide that is attached to the surface.

[0070] For instance, as depicted in FIG. 11, a first complementary sequence can include a specific nucleotide * in the region derived from the capture oligo 3' domain, and that is the only occurrence of that specific nucleotide in the region derived from the capture oligo 3' domain. The second complementary sequence includes the complement nucleotide *' atIP-2912-PCT / 0531.002912WOO 1the same position. The second capture oligo 24 on the surface invades the double stranded structure of attached first complementary sequence 32 and hybridized unattached second complementary sequence 35, and the 3' end of the second capture oligo 24 is extended 40 to result in an extended second oligo 41. When the mixture of dNTPs includes at least one labeled dNTP and does not include the dNTP complement of nucleotide extension cannot proceed beyond nucleotide The resulting double stranded structure of second complementary sequence 35 and extended second capture oligo 41 can be the substrate for another single stranded invasion by second capture oligo 24. As a result, after multiple rounds of recombinase-facilitated amplification the surface includes multiple copies of extended second capture oligo 41 that are evenly labeled, regardless of the RNA molecule that was originally used to produce the second complementary sequence 35.

[0071] Measuring label associated with a surface

[0072] After recombinase-facilitated amplification, the amount of label incorporated during the amplification is determined. It is this step that allows identification of suitable permeabilization conditions for a tissue. Suitable permeabilization conditions result in release of polynucleotides from tissue to allow the polynucleotides to diffuse and interact with capture oligos. However, too much permeabilization can result in increased diffusion and reduced interaction between polynucleotides and capture oligos. Different permeabilization conditions can be evaluated by identifying the amount of label associated with a surface to which a tissue had been applied. In general, the permeabilization conditions for a tissue resulting in the greatest amount of label associated with a surface are the conditions that are most desirable for use in subsequent experiments with that particular type of tissue.

[0073] Any method for measuring label associated with a surface can be used. In one embodiment, the label is measured by imaging (e.g., by observation with a microscope, see Example 2). This permits identification of specific areas on the surface where a tissue had been applied, and may be useful when spatial sequencing libraries of a specific part of a tissue are desired. Moreover, the relative abundance of the label can be used to determine the transcriptional activity of different regions of a tissue sample, e.g., to identify cells withIP-2912-PCT / 0531.002912WOO 1high or no transcriptional activity. Tn another embodiment, the total amount of label present on a surface to which a tissue had been applied can be determined. In this aspect, the capture oligos used can include a cleavage site that allows removal of all labeled nucleotides associated with a surface. After removal, the resulting mixture of total label can be determined by, for instance, a plate reader or slide scanner.

[0074] Compositions

[0075] The present disclosure includes compositions that can result during the practice of the methods described herein. Examples of compositions include those depicted in any of FIGs. 9-12. Another example of a composition includes an amplification reagent that is useful in producing the compositions shown in FIGs. 11-12. Such amplification reagents are missing one dNTP, e.g., they include one dNTP, two dNTPs, or three dNTPS, and one of the dNTPs is labeled.

[0076] Kits

[0077] The present disclosure also provides kits for identifying permeabilization conditions and for producing sequencing libraries. A kit can include a plurality of surfaces that include co-grafted capture oligos. One or both populations of attached capture oligos can include a cleavage site. One or both populations can include a nucleotide sequence that, when paired with a mixture of one, two, or three dNTPs, causes the termination of extension (see, for instance, FIG. 11). Examples of other components in a kit include positive control polynucleotides and / or negative control polynucleotides. Optionally, other reagents such as buffers and solutions needed to use the surfaces in the methods described herein are also included. Instructions for use of the packaged components are also typically included.

[0078] As used herein, the phrase "packaging material" refers to one or more physical structures used to house the contents of the kit. The packaging material is constructed by known methods, preferably to provide a sterile, contaminant-free environment. The packaging material has a label which indicates that the components can be used for producing sequencing libraries. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to practice reaction with a transposomeIP-2912-PCT / 0531.002912WOO 1complex and how to use template particles to form pre-templated instant partitions. As used herein, the term "package" refers to a solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding within fixed limits the components. "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter, such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent / sample admixtures, temperature, buffer conditions, and the like.

[0079] Terms used herein will be understood to take on their ordinary meaning in the relevant art unless specified otherwise. Several terms used herein and their meanings are set forth below.

[0080] As used herein, the term “amplicon,” when used in reference to a nucleic acid, means the product of copying the nucleic acid, where the product has a nucleotide sequence that is the same as or complementary to at least a portion of the nucleotide sequence of the nucleic acid. An amplicon can be produced by any of a variety of amplification methods that use the nucleic acid, e.g., a target polynucleotide or an amplicon thereof, as a template including, for example, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation extension, or ligation chain reaction. An amplicon can be a nucleic acid molecule having a single copy of a particular nucleotide sequence (e.g., a polymerase extension product) or multiple copies of the nucleotide sequence (e.g., a concatemeric product of RCA). A first amplicon of a target polynucleotide is typically a complementary copy. Subsequent amplicons are copies that are created, after generation of the first amplicon, from the target polynucleotide or from the first amplicon. A subsequent amplicon can have a sequence that is substantially complementary to the target polynucleotide or substantially identical to the target polynucleotide.

[0081] As used herein, the terms “polynucleotide” and “nucleic acid” are used interchangeably and are intended to be consistent with their use in the art and includes naturally occurring polynucleotides and functional analogs thereof. The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise. For clarity of description the terms can be used to distinguish oneIP-2912-PCT / 0531.002912WOO 1species of nucleic acid from another when describing a particular method or composition that includes several nucleic acid species. Particularly useful functional analogs are capable of hybridizing to a nucleic acid in a sequence specific fashion or capable of being used as a template for replication of a particular nucleotide sequence. Naturally occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)). A nucleic acid can contain any of a variety of analogs of these sugar moi eties that are known in the art. A nucleic acid can include native or non-native bases. In this regard, a native deoxyribonucleic acid can have one or more bases selected from adenine, thymine, cytosine or guanine and a ribonucleic acid can have one or more bases selected from uracil, adenine, cytosine or guanine. Useful non-native bases that can be included in a nucleic acid are known in the art. The term “target,” when used in reference to a polynucleotide, is intended as a semantic identifier for the polynucleotide in the context of a method or composition set forth herein and does not necessarily limit the structure or function of the polynucleotide beyond what is otherwise explicitly indicated.

[0082] As used herein, the term “polymerase” is intended to be consistent with its use in the art and includes, for example, an enzyme that produces a complementary replicate of a nucleic acid molecule using the nucleic acid as a template strand. Typically, polymerases bind to the template strand and then move down the template strand sequentially adding nucleotides to the free hydroxyl group at the 3' end of a growing strand of nucleic acid. DNA polymerases typically synthesize complementary DNA molecules from DNA templates and RNA-dependent DNA polymerases (e.g., reverse transcriptases) typically synthesize DNA molecules from RNA templates. Polymerases can use a short DNA strand, called a primer, to begin strand growth. Some polymerases can displace the strand upstream of the site where they are adding bases to a chain. Such polymerases are said to be strand displacing, meaning they have an activity that removes a complementary strand from a template strand being read by the polymerase. Exemplary polymerases having strand displacing activity include, without limitation, the large fragment of Bsu (Bacillus subtilis), Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase orIP-2912-PCT / 0531.002912WOO 1sequencing grade T7 exo-polymerase. Some polymerases degrade the strand in front of them, effectively replacing it with the growing chain behind (5' exonuclease activity). Some polymerases have an activity that degrades the strand behind them (3' exonuclease activity). Some useful polymerases have been modified, either by mutation or otherwise, to reduce or eliminate 3' and / or 5' exonuclease activity.

[0083] As used herein, "amplify", "amplifying" or "amplification reaction" and their derivatives, refer generally to any action or process whereby at least a portion of a nucleic acid molecule is replicated or copied into at least one additional nucleic acid molecule. The additional nucleic acid molecule optionally includes sequence that is substantially identical or substantially complementary to at least some portion of the template nucleic acid molecule. The template nucleic acid molecule can be single-stranded or double-stranded and the additional nucleic acid molecule can independently be single-stranded or doublestranded. Amplification is the linear or exponential replication of a nucleic acid molecule. In some embodiments, such amplification can be performed using isothermal conditions; in other embodiments, such amplification can include thermocycling. In some embodiments, the amplification is a multiplex amplification that includes the simultaneous amplification of a plurality of target sequences in a single amplification reaction. In some embodiments, "amplification" includes amplification of at least some portion of DNA and RNA based nucleic acids alone, or in combination. The amplification reaction can include any of the amplification processes known to one of ordinary skill in the art. In some embodiments, the amplification reaction includes polymerase chain reaction (PCR).

[0084] As used herein, the term "universal," when used to describe a nucleotide sequence, refers to a region of sequence that is common to two or more nucleic acid molecules where the molecules also have regions of sequence that differ from each other. A universal sequence that is present in different members of a collection of nucleic acids can be used as, for instance, a "landing pad" in a subsequent step to anneal a nucleotide sequence that can be used as a primer for addition of another nucleotide sequence, such as a universal sequence, to a target nucleic acid. A universal sequence that is present in different members of a collection of nucleic acids can allow capture of multiple different nucleic acids using a population of capture nucleic acids, e.g., capture oligonucleotides that are complementaryIP-2912-PCT / 0531.002912WOO 1to a portion of the universal sequence, e.g., a universal capture sequence. Non-limiting examples of universal capture sequences include sequences that are identical to or complementary to P5 and P7 primers. Similarly, a universal sequence present in different members of a collection of molecules can allow the replication (e.g., sequencing) or amplification of multiple different nucleic acids using a population of universal primers that are complementary to a portion of the universal sequence, e.g., a universal anchor sequence. In one embodiment universal anchor sequences are used as a site to which a universal primer (e.g., a sequencing primer for read 1 or read 2) anneals for sequencing. A capture oligo or a universal primer can therefore include a sequence that can hybridize specifically to a universal sequence.

[0085] As used herein, a "biological sample" is a sample obtained from a subject and may include one or more biological or chemical substances, such as polynucleotides, proteins, cells, tissues, organisms, and / or biologically active chemical compound(s), such as analogs or mimetics of the aforementioned species.

[0086] As used herein, the terms "organism," "subject," are used interchangeably and refer to a microbe (e.g., prokaryotic or eukaryotic), an animal, and a plant. An example of an animal is a mammal, such as a human.

[0087] As used herein, "tissue" refers to an aggregation of cells, and optionally, intercellular matter. Typically, the cells in a tissue are not free floating in solution and instead are attached to each other to form a multicellular structure. Exemplary tissue types include, but are not limited to, muscle, nerve, epidermal and connective tissues. In some instances, the biological sample may include whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardia, fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, microbes including microbial pathogens, viruses including viral pathogens, liquids containing multi-celled organisms, biological swabs and biological washes. In further examples, the sample can be derived from an organ, including for example, anIP-2912-PCT / 0531.002912WOO 1organ of the musculoskeletal system such as muscle, bone, tendon or ligament; an organ of the digestive system such as salivary gland, pharynx, esophagus, stomach, small intestine, large intestine, liver, gallbladder or pancreas; an organ of the respiratory system such as larynx, trachea, bronchi, lungs or diaphragm; an organ of the urinary system such as kidney, ureter, bladder or urethra; a reproductive organ such as ovary, fallopian tube, uterus, vagina, placenta, testicle, epididymis, vas deferens, seminal vesicle, prostate, penis or scrotum; an organ of the endocrine system such as pituitary gland, pineal gland, thyroid gland, parathyroid gland, or adrenal gland; an organ of the circulatory system such as heart, artery, vein or capillary; an organ of the lymphatic system such as lymphatic vessel, lymph node, bone marrow, thymus or spleen; an organ of the central nervous system such as brain, brainstem, cerebellum, spinal cord, cranial nerve, or spinal nerve; a sensory organ such as eye, ear, nose, or tongue; or an organ of the integument such as skin, subcutaneous tissue or mammary gland. In various embodiments, the tissue can be derived from a multicellular organism. The tissue can be freshly excised from an organism, or it may have been previously preserved for example by freezing (e.g., fresh frozen tissue), embedding in a material such as paraffin (e.g., formalin fixed paraffin embedded (FFPE) samples), formalin fixation, infiltration, dehydration or the like.

[0088] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0089] As used in this specification and the appended claims, the term "or" is generally employed in its sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. The use of "and / or" in some instances does not imply that the use of "or" in other instances may not mean "and / or."

[0090] The words "preferred" and "preferably" refer to embodiments of the disclousre that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the disclosure.IP-2912-PCT / 0531.002912WOO 1

[0091] As used herein, "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" or the like are used in their open ended inclusive sense, and generally mean "include, but not limited to," "includes, but not limited to," or "including, but not limited to."

[0092] It is understood that wherever embodiments are described herein with the language "have,""has," "having," "include," "includes," "including," "comprise," "comprises," "comprising" and the like, otherwise analogous embodiments described in terms of "consisting of and / or "consisting essentially of are also provided. The term "consisting of means including, and limited to, whatever follows the phrase "consisting of." That is, "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. The term "consisting essentially of indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.

[0093] Conditions that are "suitable" for an event to occur or "suitable" conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and / or are conducive to the event.

[0094] As used herein, "providing" in the context of, for instance, a composition, an article, or a tissue, means making the composition, the article, or the tissue, purchasing the composition, the article, or the tissue, or otherwise obtaining the composition, the article, or the tissue.

[0095] Reference throughout this specification to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.IP-2912-PCT / 0531.002912WOO 1

[0096] Throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0097] In the description herein particular embodiments may be described in isolation for clarity.Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.

[0098] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0099] The above summary of the present disclosure is not intended to describe each disclosed embodiment or every implementation of the present disclosure. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0100] The invention is defined in the claims. However, below there is provided a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.IP-2912-PCT / 0531.002912WOO 1

[0101] Exemplary Aspects

[0102] Aspect 1. A method for accessing tissue permeabilization, comprising: providing a surface comprising a plurality of immobilized capture oligos, wherein the capture oligos are immobilized at the 5’ end, wherein the capture oligos comprise a 3’ end region; contacting the surface with a tissue comprising a plurality of mRNA molecules in cells; permeabilizing the tissue to release mRNA molecules from the cells; wherein a region of the mRNA molecules anneals to the 3’ end region of the capture oligos, using reverse transcriptase (RT) to extend the 3’ end of the capture oligos using the annealed mRNA molecules as template to produce a first complementary sequence, wherein the RT adds non-templated nucleotides to the 3’ end of the first complementary sequence; annealing a template switch oligo (TSO) to the non-templated nucleotides; extending the 3’ end of the non-templated nucleotides using the 5’ end of the TSO as a template to result in a complementary TSO that is attached to the non-templated nucleotides and the first complementary sequence; annealing the TSO to the complementary TSO; extending the TSO using the non-templated nucleotides and the first complementary sequence as a template to result in a second complementary sequence; amplifying at least a portion of the second complementary sequence in the presence of a labeled dNTP to result in labeled polynucleotides immobilized to the surface; and determining the amount of the labeled dNTP associated with the surface.

[0103] Aspect 2. The method of any of aspects 1 or 3-21, wherein the surface is glass, plastic, or open-wafer.

[0104] Aspect 3. The method of any of aspects 1-2 or 4-21, wherein the capture oligos are from 20 to 50 nucleotides in length.

[0105] Aspect 4. The method of any of aspects 1-3 or 5-21, wherein the capture oligos comprise a cleavage site.

[0106] Aspect 5. The method of any of aspects 1-4 or 6-21, wherein the cleavage site comprises a restriction endonuclease site, a deoxyuridine, a photocleavable linker, a diol linkage, or a disulfide group.IP-2912-PCT / 0531.002912WOO 1

[0107] Aspect 6. The method of any of aspects 1-5 or 7-21, wherein the 3’ end region comprises a poly-T domain, a random er, a target domain, or a disrupted homopolymer.

[0108] Aspect 7. The method of any of aspects 1-6 or 8-21, wherein the permeabilizing comprises exposing the tissue to a permeabilization agent.

[0109] Aspect 8. The method of any of aspects 1-7 or 9-21, wherein the permeabilization agent comprises an organic solvent or a detergent.

[0110] Aspect 9. The method of any of aspects 1-8 or 10-21, wherein the organic solvent comprises methanol or acetone.

[0111] Aspect 10. The method of any of aspects 1-9 or 11-21, wherein the detergent comprises NP40, streptolysin O, a saponin, Triton X-100, or Tween-20.

[0112] Aspect 11. The method of any of aspects 1-10- or 12-21, wherein the RT comprises a Moloney murine leukemia virus RT.

[0113] Aspect 12. The method of any of aspects 1-11 or 13-21, wherein the non-templated nucleotides comprise dCTP.

[0114] Aspect 13. The metho of any of aspects 1-12 or 14-21, wherein the amplifying comprises strand invasion.

[0115] Aspect 14. The method of any of aspects 1-13 or 15-21, wherein the amplifying comprises kinetic exclusion amplification.

[0116] Aspect 15. The method of any of aspects 1-14 or 16-21, wherein the labeled dNTP comprises a fluorescent moiety, biotin, peptide, or azide moiety.

[0117] Aspect 16. The method of any of aspects 1-15 or 17-21, wherein the amplifying comprises a subset of dNTP such that only a part of the second complementary sequence is amplified in the presence of the subset of dNTP to result in truncated labeled polynucleotides immobilized to the surface.IP-2912-PCT / 0531.002912WOO 1

[0118] Aspect 17. The method of any of aspects 1-16 or 18-21, wherein the determining comprises identifying the label by microscopy.

[0119] Aspect 18. The method of any of aspects 1-17 or 19-21, wherein the determining comprises identifying the label by fluorescent imaging.

[0120] Aspect 19. The method of any of aspects 1-18 or 20-21, wherein the fluorescent imaging is fluorescence microscopy.

[0121] Aspect 20. The method of any of aspects 1-19 or 21, wherein the determining comprises identifying the label using a fluorescent plate reader or fluorescence slide scanner.

[0122] Aspect 21. The method of aspect 1, wherein the determining comprises cleaving the labeled polynucleotides from the surface and measuring the amount of label associated with the cleaved labeled polynucleotides.

[0123] Aspect 22. A method for accessing tissue permeabilization, comprising: providing a surface comprising immobilized capture oligos, wherein the capture oligos are immobilized at the 5’ end, wherein the capture oligos comprise a 3’ end region; contacting the surface with a tissue comprising mRNA molecules in cells; permeabilizing the tissue to release mRNA molecules from the cells; wherein a region of the mRNA molecules anneals to the 3’ end region of the capture oligos, using reverse transcriptase (RT) to extend the 3’ end of the capture oligos using the annealed mRNA molecules as template to produce a first complementary sequence, annealing a primer to nucleotides of the first complementary sequence, extending the primer 3’ end using the first complementary sequence as a template to result in a second complementary sequence; amplifying at least a portion of the second complementary sequence in the presence of a labeled dNTP to result in labeled polynucleotides immobilized to the surface; and determining the amount of the labeled dNTP associated with the surface.

[0124] Aspect 23. The method of any of aspects 22- or 24-43, wherein the surface is glass, plastic, or open-wafer.IP-2912-PCT / 0531.002912WOO 1

[0125] Aspect 24. The method of any of aspects 22-23 or 25-43, wherein the capture oligos are from 20 to 50 nucleotides in length.

[0126] Aspect 25. The method of any of aspects 22-24 or 26-43, wherein the capture oligos comprise a cleavage site.

[0127] Aspect 26. The method of any of aspects 22-25 or 27-43, wherein the cleavage site comprises a restriction endonuclease site, a deoxyuridine, a photocleavable linker, a diol linkage, or a disulfide group.

[0128] Aspect 27. The method of any of aspects 22-26 or 28-43, wherein the 3’ end region comprises a poly-T domain, a randomer, a target domain, or a disrupted homopolymer.

[0129] Aspect 28. The method of any of aspects 22-27 or 29-43, wherein the permeabilizing comprises exposing the tissue to a permeabilization agent.

[0130] Aspect 29. The method of any of aspects 22-28 or 30-43, wherein the permeabilization agent comprises an organic solvent or a detergent.

[0131] Aspect 30. The method of any of aspects 22-29 or 31-43, wherein the organic solvent comprises methanol or acetone.

[0132] Aspect 31. The method of any of aspects 22-30 or 32-43, wherein the detergent comprises NP40, streptolysin O, a saponin, Triton X-100, or Tween-20.

[0133] Aspect 32. The method of any of aspects 22-31 or 33-43, wherein the RT comprises a Moloney murine leukemia virus RT.

[0134] Aspect 33. The method of any of aspects 22-32 or 34-43, wherein the primer anneals to a pre-determined sequence of the first complementary sequence.

[0135] Aspect 34. The method of any of aspects 22-33 or 35-43, wherein the primer comprises a random sequence of nucleotides.

[0136] Aspect 35. The method of any of aspects 22-34 or 36-43, wherein the amplifying comprises strand invasion.IP-2912-PCT / 0531.002912WOO 1

[0137] Aspect 36. The method of any of aspects 22-35 or 37-43, wherein the amplifying comprises kinetic exclusion amplification.

[0138] Aspect 37. The method of any of aspects 22-36 or 38-43, wherein the labeled dNTP comprises a fluorescent moiety, biotin, peptide, or azide moiety.

[0139] Aspect 38. The method of any of aspects 22-37 or 39-43, wherein the amplifying comprises a subset of dNTP such that only a part of the second complementary sequence is amplified in the presence of the subset of dNTP to result in truncated labeled polynucleotides immobilized to the surface.

[0140] Aspect 39. The method of any of aspects 22-38 or 40-43, wherein the determining comprises identifying the label by microscopy.

[0141] Aspect 40. The method of any of aspects 22-39 or 41-43, wherein the determining comprises identifying the label by fluorescent imaging.

[0142] Aspect 41. The method of any of aspects 22- 40 or 42-43, wherein the fluorescent imaging is fluorescence microscopy.

[0143] Aspect 42. The method of any of aspects 22- 41 or 43, wherein the determining comprises identifying the label using a fluorescent plate reader or fluorescence slide scanner.

[0144] Aspect 43. The method of any of aspects 22-42, wherein the determining comprises cleaving the labeled polynucleotides from the surface and measuring the amount of label associated with the cleaved labeled polynucleotides.

[0145]

[0146] EXAMPLES

[0147] The present disclosure is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the disclosure as set forth herein.IP-2912-PCT / 0531.002912WOO 1

[0148] Example 1

[0149] Proof-of-Concept Tissue-Free Assay

[0150] To test the feasibility of the assay, flowcells in an 8-lane format were grafted to screen different conditions such as changing oligo graft input concentration, number of grafted oligos, and input of the label, in this case a Cy3-dCTP. Cy3-dCTP was added to ExAmp mix after reverse transcription (RT) (with total RNA) and mRNA removal to yield a fluorescent signal and imaged by a flatbed scanner in the Cy3 channel.

[0151] Flowcells were grafted with a single capture oligo (see FIG. 2A for substrate design) or cografted with two capture oligos (see FIG. 2B for substrate design) and tested using a cBot as a fluidic device to deliver reagents. Mouse kidney total RNA was used as the source of mRNA for reverse transcription (RT). SBS3 template switch oligo (TSO) was incorporated during RT, followed by mRNA removal and second strand synthesis off the SBS3 TSO. Cy3-dCTP-spiked ExAmp was flowed in for 1 h at 38 °C and the flowcells aggressively washed at 60°C in low salt buffer. As shown in FIG. 13, co-grafted lanes at -85-120 pM Cy3-dCTP had the highest RFU signal, comparable to the positive control. Thus, results showed that co-grafted surfaces outperformed the single graft.

[0152] Example 2

[0153] Demonstration of Assay Performance

[0154] Permeabilization experiments were performed using cryosections of mouse kidney (FIG.14), mouse cerebellum (FIG. 15) and mouse heart (FIG. 16) and demonstrated the usage of the ExAmp method to determine useful permeabilization times. A cryostat was used to serially section the tissues and mount them onto the “co-grafted” flowcells (see Example 1). The sections were fixed with methanol and stained with Hemotoxylin and Eosin. The sections were then permeabilized over different times (see FIGs. 14-16) and reverse transcription was performed in the presence of SBS3-TSO. After reverse transcription, tissue digestion, RNA removal, second strand synthesis with a primer complementary to a portion of SB S3 -TSO were performed. The surface was washed and ExAmp performedIP-2912-PCT / 0531.002912WOO 1with Cy3-dCTP for 30 minutes. After washing the surfaces were imaged with a fluorescence microscope.

[0155] For mouse kidney (FIG. 14), fluorescence images of the mouse kidney sections after 2 minutes, 7 minutes, 12 minutes, 17 minutes, 30 minutes of permeabilization were used to determine a useful permeabilization time. The images (presented here with same brightness and contrast settings) show that there is an increase in intensity from 2 minutes to 7 minutes; intensity remains the same for 7 minutes and 12 minutes and then the images get less intense from 17 minutes to 30 minutes. Analysis of these images indicates a useful permeabilization time for mouse kidney to be around 7 minutes- 12 minutes.

[0156] For mouse cerebellum (FIG. 15), fluorescence images of the mouse cerebellum sections after 2 minutes, 5 minutes, 10 minutes, 15 minutes, 25 minutes of permeabilization can be used to determine a useful permeabilization time. The images (presented here with same brightness and contrast settings) show that there is an increase in intensity from 2 minutes to 5 minutes to 10 minutes and then the images get less intense from 10 minutes to 15 minutes. The intensity seems to increase for the 25 minutes time point, but the image appears blurry and diffuse. Analysis of these images indicates a useful permeabilization time for mouse cerebellum to be around 10 minutes.

[0157] For mouse heart (FIG. 16), fluorescence images of the mouse heart sections after 5 minutes, 15 minutes, 27 minutes, 37 minutes, 45 minutes of permeabilization can be used to determine a useful permeabilization time. The images (presented here with same brightness and contrast settings) show that the intensity is highest for the 5 minutes time point and then the intensity decreases as we go to longer and longer points. Analysis of these images indicates a useful permeabilization time for mouse heart to be around 5 minutes.

[0158] Incorporation by Reference

[0159] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt,IP-2912-PCT / 0531.002912WOO 1PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. Supplementary materials referenced in publications (such as supplementary tables, supplementary figures, supplementary materials and methods, and / or supplementary experimental data) are likewise incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure / s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

[0160] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0161] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0162] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

PCT / US25 / 59803 16 December 2025 (16.12.2025)IP-2912-PCT / 0531.002912WOO 1CLAIMS1. A method for accessing tissue permeabilization, comprising:providing a surface comprising a plurality of immobilized capture oligos,wherein the capture oligos are immobilized at the 5’ end,wherein the capture oligos comprise a 3’ end region;contacting the surface with a tissue comprising a plurality of mRNA molecules in cells;permeabilizing the tissue to release mRNA molecules from the cells;wherein a region of the mRNA molecules anneals to the 3’ end region of the capture oligos,using reverse transcriptase (RT) to extend the 3 ’ end of the capture oligos using the annealed mRNA molecules as template to produce a first complementary sequence,wherein the RT adds non-templated nucleotides to the 3’ end of the first complementary sequence;annealing a template switch oligo (TSO) to the non-templated nucleotides;extending the 3’ end of the non-templated nucleotides using the 5’ end of the TSO as a template to result in a complementary TSO that is attached to the non-templated nucleotides and the first complementary sequence;annealing the TSO to the complementary TSO;extending the TSO using the non-templated nucleotides and the first complementary sequence as a template to result in a second complementary sequence;amplifying at least a portion of the second complementary sequence in the presence of a labeled dNTP to result in labeled polynucleotides immobilized to the surface; andPCT / US25 / 59803 16 December 2025 (16.12.2025)IP-2912-PCT / 0531.002912WOO 1determining the amount of the labeled dNTP associated with the surface.

2. A method for accessing tissue permeabilization, comprising:providing a surface comprising immobilized capture oligos,wherein the capture oligos are immobilized at the 5’ end,wherein the capture oligos comprise a 3’ end region;contacting the surface with a tissue comprising mRNA molecules in cells;permeabilizing the tissue to release mRNA molecules from the cells;wherein a region of the mRNA molecules anneals to the 3’ end region of the capture oligos,using reverse transcriptase (RT) to extend the 3 ’ end of the capture oligos using the annealed mRNA molecules as template to produce a first complementary sequence;annealing a primer to nucleotides of the first complementary sequence,extending the primer 3’ end using the first complementary sequence as a template to result in a second complementary sequence;amplifying at least a portion of the second complementary sequence in the presence of a labeled dNTP to result in labeled polynucleotides immobilized to the surface; anddetermining the amount of the labeled dNTP associated with the surface.

3. The method of claim 1 or 2, wherein the surface is glass, plastic, or open-wafer.

4. The method of claim 1 or 2, wherein the capture oligos are from 20 to 50 nucleotides in length.

5. The method of claim 1 or 2, wherein the capture oligos comprise a cleavage site.PCT / US25 / 59803 16 December 2025 (16.12.2025)IP-2912-PCT / 0531.002912WOO 16. The method of claim 5, wherein the cleavage site comprises a restriction endonuclease site, a deoxyuridine, a photocleavable linker, a diol linkage, or a disulfide group.

7. The method of claim 1 or 2, wherein the 3’ end region comprises a poly-T domain, a randomer, a target domain, or a disrupted homopolymer.

8. The method of claim 1 or 2, wherein the permeabilizing comprises exposing the tissue to a permeabilization agent.

9. The method of claim 8, wherein the permeabilization agent comprises an organic solvent or a detergent.

10. The method of claim 9, wherein the organic solvent comprises methanol or acetone.

11. The method of claim 9, wherein the detergent comprises NP40, streptolysin 0, a saponin, Triton X-100, or Tween-20.

12. The method of claim 1 or 2, wherein the RT comprises a Moloney murine leukemia virus RT.

13. The method of claim 1, wherein the non-templated nucleotides comprise dCTP.

14. The method of claim 2, wherein the primer anneals to a pre-determined sequence of the first complementary sequence.

15. The method of claim 2, wherein the primer comprises a random sequence of nucleotides.

16. The method of claim 1 or 2, wherein the amplifying comprises strand invasion.

17. The method of claim 16, wherein the amplifying comprises kinetic exclusion amplification.

18. The method of claim 1 or 2, wherein the labeled dNTP comprises a fluorescent moiety, biotin, peptide, or azide moiety.PCT / US25 / 59803 16 December 2025 (16.12.2025)IP-2912-PCT / 0531.002912WOO 119. The method of claim 1 or 2, wherein the amplifying comprises a subset of dNTP such that only a part of the second complementary sequence is amplified in the presence of the subset of dNTP to result in truncated labeled polynucleotides immobilized to the surface.

20. The method of claim 1 or 2, wherein the determining comprises identifying the label by microscopy.

21. The method of claim 1 or 2, wherein the determining comprises identifying the label by fluorescent imaging.

22. The method of claim 21, wherein the fluorescent imaging is fluorescence microscopy.

23. The method of claim 1 or 2, wherein the determining comprises identifying the label using a fluorescent plate reader or fluorescence slide scanner.

24. The method of claim 1 or 2, wherein the determining comprises cleaving the labeled polynucleotides from the surface and measuring the amount of label associated with the cleaved labeled polynucleotides.

Images