Methods and compositions for identifying 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 cells, such as cells present in a tissue, to release biological material for subsequent interaction with immobilized capture probes.

Description

IP-2924-PCT / 531.2924WO01METHODS AND COMPOSITIONS FOR IDENTIFYING PERMEABILIZATION CONDITIONS FOR PRODUCING SPATIAL SEQUENCING LIBRARIES

[0001] FIELD

[0002] 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 cells associated with a surface to release analytes for subsequent interaction with immobilized capture probes.

[0003] BACKGROUND

[0004] Spatial transcriptomics enables highly multiplexed in situ gene expression profding 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 analytes, such as mRNA or protein, are released and subsequently captured on the surface. Current spatial workflows, however, are inefficient at capturing analytes, which reduces sensitivity. For instance, less than 1% mRNA is typically captured and converted within a tissue section.

[0005] SUMMARY OF THE APPLICATION

[0006] Provided herein are surfaces with attached capture probes. In one embodiment, the capture probes are attached to the surface at the 5’ ends. In one embodiment, members of a first population of the attached capture probes can include a 3’ end that is covalently attached to a first complementary strand having a nucleotide sequence complementary to one of a plurality of polynucleotides, and the first complementary strands of the first population of the attached capture probes do not include a label. Members of a second population of the attached capture probes can include a 3’ end that is covalently attached to a second complementary strand that includes at least one label. In one embodiment, the second complementary strand of each member of the second population of the attached capture probes is identical.IP-2924-PCT / 531.2924WO01

[0007] In one embodiment, a surface includes a plurality of capture probes attached to the surface.In one embodiment, each of a first population of the plurality of attached capture probes can include a moiety bound to a non-polynucleotide analyte, and the attached capture probes having a moiety bound to a non-polynucleotide analyte do not include a label. In one embodiment, each of a second population of the plurality of attached capture probes include a secondary analyte, and the secondary analyte includes at least one label. In one embodiment, the secondary analyte of each member of the second population of the attached capture probes is identical.

[0008] Also provided herein are methods for identifying permeabilization conditions for a tissue or cells associated with a surface. In one embodiment, a method includes providing a surface including a plurality of attached capture probes and cells, such as a tissue, where the tissue includes tissue analytes. A method can include permeabilizing the tissue to release the tissue analytes under conditions suitable for the tissue analytes to bind to the capture probes, thereby converting the plurality of attached capture probes to a first population of occupied capture probes and a second population of unoccupied capture probes. A method can include labeling the unoccupied capture probes, and determining the amount of the labeled associated with the surface.

[0009] In one embodiment, a method included providing a surface including attached capture probes and cells, for instance a tissue, where the tissue includes a first analyte that includes polynucleotides. In one embodiment, the capture probes are attached at the 5’ end. In one embodiment, the capture probes include a 3’ capture domain. A method can include permeabilizing the tissue to release the polynucleotides, where a region of each of the polynucleotides hybridizes to the 3’ capture domain of a capture probe, thereby converting a plurality of capture probes to a first population of occupied capture probes and a second population of unoccupied capture probes. A method can include extending the 3’ end of the first population of capture probes using the hybridized polynucleotides as template to produce first complementary strands attached to the 3’ end of each first population capture probe, and hybridizing a polynucleotide second analyte to the 3’ capture domain of the second population of capture probes. In one embodiment, the polynucleotide second analyte includes a first region that hybridizes to the 3’ capture domain of the secondIP-2924-PCT / 531.2924WO01population of capture probes, and a second region at the 5’ end that does not hybridize to the 3’ capture domain of the second population of unoccupied capture probes. A method can include using the hybridized polynucleotide second analyte to attach a label to the 3’ end of each second population capture probe, and determining the amount of the labeled associated with the surface.

[0010] 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.

[0011] BRIEF DESCRIPTION OF THE FIGURES

[0012] The following detailed description of illustrative embodiments of the present disclosure may be best understood when read in conjunction with the following drawings.

[0013] FIG. 1 shows a general block diagram of a portion of a general illustrative workflow for determining permeabilization conditions.

[0014] FIG. 2 shows a general block diagram of a portion of a general illustrative workflow for determining permeabilization conditions when a non-polynucleotide analyte is being analyzed. The block outlined in a dashed line indicates a step that is optional in some embodiments.

[0015] FIG. 3A-3C shows schematic drawings of embodiments for identifying permeabilization conditions when a protein analyte is being analyzed. For simplicity, only a limited area of a surface with attached capture probes and other structures produced during practice of the methods disclosed herein are shown.

[0016] FIG. 4 shows a general block diagram of a portion of a general illustrative workflow for determining permeabilization conditions when a polynucleotide analyte is being analyzed. Blocks outlined in a dashed line indicate steps that are optional in some embodiments.IP-2924-PCT / 531.2924WO01

[0017] FTG. 5A-5K shows schematic drawings of embodiments for identifying permeabilization conditions when a polynucleotide analyte is being analyzed. For simplicity, only a limited area of a surface with attached capture probes and other structures produced during practice of the methods disclosed herein are shown.

[0018] FIG. 6 shows permeabilization results for mouse cerebellum.

[0019] 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.

[0020] DETAILED DESCRIPTION

[0021] The production of on-surface libraries requires permeabilization of cells, such as a tissue, for release of molecules such as mRNA or proteins. Multiple factors, however, can affect permeabilization. Factors that can affect permeabilization 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, tissue quality, and type of cells (e.g., cells with delicate membranes vs. cells with robust membranes). Suitable permeabilization conditions must 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 includes on-surface analyte capture by immobilized capture probes; however, unlike current methods, the first capture is followed with a second on-surface capture by unoccupied immobilized capture probes. The second on-surface capture includes label that can be used to identify suitable permeabilization conditions. In those embodiments described herein where polynucleotides from a tissue are evaluated, cDNAs are not labeled, thus, unlike current methods, the cDNAs producedIP-2924-PCT / 531.2924WO01during the identification of permeabilization conditions can be used for the construction of on-surface libraries.

[0022] Provided herein are methods, compositions, and kits for determining permeabilization conditions. In some embodiments, a workflow includes providing a surface with attached capture probes, occupying capture probes with analytes from a biological sample, labeling the unoccupied capture probes, and determining the amount of label associated with the unoccupied capture probes (FIG. 1).

[0023] Surfaces

[0024] Methods of the present disclosure can include providing a surface with attached capture probes (FIG. 1, block 10). As used herein, a “surface” refers to any material that is appropriate for, or can be modified to be appropriate for, the attachment of the capture oligos. As will be appreciated by those in the art, the number of possible surfaces is very large. Examples of substrates include, but are not limited 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, plastics, and a variety of other polymers. A substrate is generally rigid and is insoluble in an aqueous liquid.

[0025] A surface useful in the methods described herein includes any material that can serve as a solid or semi-solid foundation for creation of features such as wells for the deposition of biopolymers, including polynucleotide, protein, and / or other polymers. A substrate as provided herein is modified, for example, or can be modified to accommodate attachment of biopolymers by a variety of methods known to those skilled in the art.

[0026] A surface can be in a form suitable for application of cells, for instance 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 plate, such as a multi-well plate. A substrate can be non-patterned, e.g., substantially planar.IP-2924-PCT / 531.2924WO01Alternatively, a surface can be patterned, e ., include depressions. A patterned surface may be fabricated as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the array substrate.

[0027] A surface can include fiducial markers, i.e., objects that will be in the field of view of an imaging system and appear in an image produced. Fiducial markers are typically used as a point of reference or measurement scale. For instance, they can be used to provide orientation of tissue on the surface in relation to an attached capture probe. Fiducial markers can include, but are not limited to, labels such as fluorescent, radioactive, chemiluminescent, calorimetric, and colorimetric, and metal. The use of fiducial markers to stabilize and orient biological samples is described, for example, in Carter et al., Applied Optics 46:421-427, 2007),

[0028] Tissues and cells

[0029] A surface with attached capture probes can further include cells (FIG. 1, block 10).Methods of the present disclosure can include obtaining a tissue from a biological sample, processing the tissue, and applying the tissue to a surface. Alternatively, a plurality of cells that are not in a tissue can be applied to a surface. For example, a tissue can be dissociated to separate the cells present in the tissue and the resulting plurality of cells can be applied to a surface. In another non-limiting example, cells from a source, such as cells in a biological sample (e.g., blood) or cultured cells from a suspension culture or adherent culture, can be applied to a surface. Throughout this disclosure, reference to “tissue” encompasses both cells present in a tissue and a plurality of cells not in a tissue, unless the context indicates otherwise.

[0030] Exemplary biological samples that can be used in the methods described herein include, for example, those from a source such as an animal, for instance a mammal, such as a rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow, cat, dog, primate, human or non-human primate; a plant, such as Arabidopsis thahana, com, sorghum, oat, wheat, rice, canola, or soybean; a nematode such as Caenorhabditis ele ans an insect,IP-2924-PCT / 531.2924WO01such as Drosophila melanogaster, mosquito, fruit fly, honey bee or spider; a fish, such as zebrafish; a reptile; or an amphibian, such as a frog or Xenopus laevis. A biological sample can be obtained as a tissue sample, such as a tissue section, biopsy, or a core biopsy. A biological sample can include an organoid. A biological sample can include cells in suspension, including but not limited to, cells in a biological fluid (e.g., cells present in blood), cells obtained from a dissociated tissue, or cultured cells a suspension culture or adherent culture.

[0031] A biological sample can be obtained from any location within a source, at any developmental stage of the source. A biological sample can be homogeneous (e.g., be composed of one type of cell) or heterogenous (e.g., composed of more than one type of cell). A biological sample can include normal cells (e g., healthy) or one or more diseased cells (e.g., a tumor). A diseased cell can have altered metabolic properties, gene expression, protein expression, and / or morphologic features. Examples of diseases include inflammatory disorders, metabolic disorders, nervous system disorders, and cancer.

[0032] 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.

[0033] A tissue can be prepared by freezing at a temperature suitable for preserving the integrity of the tissue's structure and analytes. The frozen tissue can be subsequently sectioned using a low-temperature cryostat. Methods for freezing tissue and sectioning frozen tissue are known in the art. A tissue may be prepared by fixation for preserving the integrity of the tissue's structure and analytes. Fixation methods include formalin-fixation and paraffin- embedding (FFPE), immersion in ethanol, methanol, acetone, formaldehyde, paraformaldehyde-Triton, glutaraldehyde, or combinations thereof.IP-2924-PCT / 531.2924WO01

[0034] A tissue can be stained to aid in histological visualization. Any biological stain useful in identifying tissue and cellular structure can be used, including but not limited to, acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsine, hematoxylin, Hoechst stains, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranin. Known staining techniques can be used, such as Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's, and / or Periodic Acid Schiff (PAS).

[0035] In some embodiments, a biological sample is stained using one type of stain or one technique. In some embodiments, staining includes biological staining techniques such as H&E staining. In some embodiments, staining includes identifying analytes using fluorescently conjugated antibodies. In some embodiments, a biological sample is stained using two or more different types of stains or different staining techniques. For example, a tissue can be prepared by staining and imaging using one technique (e.g., H&E staining and brightfield imaging), followed by staining and imaging using another technique (e.g., immunohistochemistry / immunofluorescence staining and fluorescence microscopy) for the same tissue.

[0036] Cells, such as cells present in a tissue section, are typically applied to a surface. Any variety of characteristics can determine the transfer conditions required to apply a tissue to a surface. Examples of characteristics likely to impact transfer conditions include but are not limited to the sample (e.g., origin of the tissue, thickness, fixation, and cross-linking) and / or the analyte of interest (conditions to preserve and / or transfer different analytes (e.g., DNA, RNA, and protein)). 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. Attachment of the tissue can be irreversible or reversible, depending upon the nature of the sample and subsequent steps to be practiced.

[0037] Capture probes

[0038] A surface used in the methods of the present disclosure includes attached capture probes (FIG. 1, block 10). A capture probe includes a capture domain that can specificallyIP-2924-PCT / 531.2924WO01interact with an analyte. An analyte from cells present on a surface, such as a tissue, also referred to herein as a tissue analyte, can be a polynucleotide or non-polynucleotide.Examples of polynucleotide analytes include, but are not limited to, DNA and RNA. DNA in cells, such as cells in a tissue, includes, but is not limited to, genomic DNA, mitochondrial DNA, episomal DNA, plasmid DNA, viral DNA, and microbial DNA (e.g., from a microbial pathogen). RNA in cells, such as cells in a tissue includes, but is not limited to, RNA involved in protein synthesis (e.g., messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA)), and RNA involved in regulation (e.g., long noncoding RNA (IncRNA), microRNA (miRNA), and small interfering RNA (siRNA). A polynucleotide analyte can be single stranded, double stranded, or a hybrid that includes both single and double stranded regions. A polynucleotide analyte can be linear or circular. Examples of non-polynucleotide analytes include, but are not limited to, carbohydrates (e g., polysaccharides), lipids (e.g., fats, hormones), proteins, glycoproteins (N-linked and O-linked), and lipoproteins (e.g., high-density lipoprotein and low-density lipoprotein).

[0039] A capture domain can include polynucleotides, for instance, a series of polynucleotides that will hybridize with complementary or substantially complementary nucleotides of a polynucleotide analyte. In another embodiment, a capture probe can include a moiety that will bind to a non-polynucleotide analyte. Examples of moieties include, but are not limited to, a polynucleotide (e.g., a polynucleotide aptamer), amino acids (e.g., a protein) and ligands (e.g., a drug, a cofactor, or a hormone).

[0040] In one embodiment, a capture probe includes a capture domain having a polynucleotide that can hybridize to a single stranded polynucleotide to be analyzed (e.g., an mRNA analyte). The polynucleotide of the capture domain can be single stranded, double stranded, or have a mixture of one or more regions that are single stranded and double stranded. In some embodiments the polynucleotide of the capture domain is single stranded. The polynucleotide sequence of a capture domain can include natural nucleotides (adenine, cytosine, guanine, thymine, inosine, uracil), modified nucleotides (e.g., locked nucleic acids (LNAs), bridged nucleic acids (BNAs), peptide nucleic acids (PNAs)), or any combination thereof.IP-2924-PCT / 531.2924WO01

[0041] Examples of a polynucleotide that can hybridize to a single stranded polynucleotide analyte 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 and a disrupted homopolymer are useful in hybridizing to the poly-A region of mRNAs.

[0042] A randomer domain includes sequence of nucleotides that can exhibit fourfold degeneracy at each position. In some embodiments, a randomer domain is a random hexamer.

[0043] As used herein, a “target-specific domain,” when used in reference to a capture domain, refers to a nucleotide sequence complementary to a targeted polynucleotide analyte. For instance, nucleotides of a target-specific domain will selectively hybridize to a targeted polynucleotide, e.g., a mRNA encoded by a specific coding region. A surface can include capture probes with a target-specific domain, and optionally a surface can include different populations of capture probes, each population having a single target-specific capture domain that targets a different set of nucleotides of the same mRNA.

[0044] As used herein, a "disrupted homopolymer" refers to a capture domain 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 non-sequential nucleotides or non-sequential nucleotide sequences, where each of the non-sequential nucleotides or non-sequential nucleotide sequences is complementary to a portion of a mRNA poly-A region. Each of the non-sequential nucleotides or non-sequential 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 thymine nucleotides, followed by a non-thymine nucleotide, and a second series of three thymine nucleotides. 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 does not prevent hybridization between a mRNA poly-A region and complementary thymidine nucleotides present in a disrupted homopolymer.IP-2924-PCT / 531.2924WO01

[0045] A polynucleotide that is part of a capture domain and can hybridize to a single stranded polynucleotide is not limited by length, and can be 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides long.

[0046] A capture domain that can hybridize to a single stranded polynucleotide is typically oriented to have the 5’ end attached to the surface or anchor domain and have the 3’ end be free to interact with and hybridize to a polynucleotide (e.g., mRNA). Attachment of the capture domain in this orientation 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.

[0047] In one embodiment, a capture probe includes a capture domain that can bind to a nonpolynucleotide analyte. Such a capture domain can include, but is not limited to, a polynucleotide sequence that forms an aptamer, an amino acid sequence, e.g., a protein, or a ligand.

[0048] The polynucleotide sequence of a capture domain, e.g., a capture domain that hybridizes to a polynucleotide analyte or a capture domain that forms an aptamer, can include natural nucleotides (adenine, cytosine, guanine, thymine, inosine, uracil), modified nucleotides (e.g., locked nucleic acids (LNAs), bridged nucleic acids (BNAs), peptide nucleic acids (PNAs)), or any combination thereof.

[0049] The amino acid sequence of a capture domain can include natural amino acids, unnatural amino acids, or a combination thereof. Examples of synthetic amino acids include, but are not limited to, beta-amino acids, gamma-amino acids, and hydroxylated amino acids.

[0050] In some embodiments, the amino acid sequence of a capture domain can include an antibody. An "antibody" is a protein that recognizes and specifically binds to an antigen. An antibody that can “specifically bind” an antigen includes an antibody that interacts only with the epitope of the antigen that induced the synthesis of the antibody, or interacts with a structurally related epitope. An antibody that “specifically binds” to an epitope will, under the appropriate conditions, interact with the epitope even in the presence of a diversity ofIP-2924-PCT / 531.2924WO01potential binding targets. As used herein, the term “protein: antibody complex” refers to the complex that results when an antibody specifically binds to a protein.

[0051] The antibody of a capture domain can be polyclonal or monoclonal, and in some embodiments is monoclonal. The antibody of a capture domain can be naturally-occurring or synthetic, and include single domain antibodies (e.g. variable heavy domain of heavy chain (VHH) domains and variable new antigen receptor (VNAR) domains), single-chain fragment variable (scFv) antibody, and fragment antigen-binding (Fab) domains).

[0052] A capture probe can include an optional anchor domain. An anchor domain is attached to a surface. An anchor domain can include, but is not limited to, nucleotides, amino acids, or a combination thereof. The attachment of a capture probe to a surface, whether an anchor domain is present or not, can be covalent or non-covalent, and in one embodiment is covalent.

[0053] An anchor domain can be useful in different aspects of the methods disclosed herein. For instance, nucleotide sequences of an anchor domain can provide a universal sequence. A universal sequence can be used as a primer binding site, for instance for a sequencing reaction. A universal sequence can be used in aiding amplification, for instance, an amplification that includes strand invasion.

[0054] A capture probe 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 of a polynucleotide anchor domain 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.IP-2924-PCT / 531.2924WO01

[0055] Suitable cleavage techniques of a polynucleotide anchor domain 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.

[0056] Chemical cleavage encompasses any method that 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).

[0057] 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.

[0058] 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 predetermined 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.

[0059] 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 (NEBIP-2924-PCT / 531.2924WO01M55O5S) is used for the creation of a single nucleotide gap at a uracil base in a capture nucleic acid.

[0060] A surface can include one type of capture probe, e.g., the surface includes a homogeneous lawn or field of the same capture probe. A surface having a single population of capture probe attached is referred to herein as a grafted surface. A surface can include two or more capture probes, e.g., the surface includes a heterogeneous lawn or field of two or more capture probes. A surface having two or more populations of capture probes attached is referred to herein as a co-grafted surface. When more than one population of capture probes is present on a surface, each population of capture probe can bind to polynucleotide analytes, each population of capture probe can bind to non-polynucleotide analytes, or some populations of capture probes can bind polynucleotide analytes, and some populations of capture probes can bind non-polynucleotide analytes.

[0061] A surface can have a capture probe 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 probes per square centimeter (cm2).

[0062] A capture probe that includes nucleotides can be 8 to 80 nucleotides in length. In some embodiments, a capture 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.

[0063] A capture probe that includes amino acids can be the minimum number of amino acids needed to specifically bind an antigen. For instance, examples of scFvs can be about 25 amino acids in length, and a complete antibody molecule, e.g., two light chains and two heavy chains, can have a total of 1,320 amino acids or more. A capture probe that includes a ligand to bind a non-polynucleotide analyte can include 1, 2, 3, 4, or more copies of the ligand.IP-2924-PCT / 531.2924WO01

[0064] Optionally, a capture probe can include a barcode, a spatial barcode, a unique molecular identifier (UMI), or a combination thereof. In one embodiment, a barcode, a spatial barcode, and / or a UMI can be present in an anchor domain. In one embodiment, a capture probe does not include a barcode, does not include a UMI, or does not include both barcode and UMI. Barcodes, spatial barcodes, and UMIs can be a nucleotide sequence, thus a capture probe that includes a moiety that will bind to a non-polynucleotide analyte can also include a polynucleotide that includes the sequence of barcode, spatial barcode, and / or UMI.

[0065] Permeabilization

[0066] Methods of the present disclosure include permeabilizing the cells present on a surface, such as cells present in a tissue (FIG. 1, block 20). As used herein, “permeabilizing” the cells, such as cells present in a tissue, refers to releasing analytes present in a tissue, e.g., intracellular and extracellular analytes, and subsequent capture of analytes, e.g., polynucleotides or proteins, by capture probes attached to a surface. Examples of polynucleotides released from tissue include, but are not limited to, mRNA, gDNA, rRNA, tRNA, or a combination thereof. Examples of proteins released from tissue include, but are not limited to, extracellular proteins, intracellular proteins, cytoplasmic proteins, nuclear proteins, or a combination thereof.

[0067] Typically, conditions useful for permeabilizing one type of tissue or one type of cell do not work effectively with other tissues or cells, thus the permeabilizing can include identifying conditions that release analytes from a tissue or cells and permit capture of the analytes by the surface capture probes. Identifying conditions for permeabilizing tissue or cells 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.

[0068] 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, butIP-2924-PCT / 531.2924WO01are 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).

[0069] 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%.

[0070] 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, 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, or 60 minutes.

[0071] The temperature of the treatment can vary from at least -20 °C, at least -10 °C, at least 0 °C, 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.

[0072] In some embodiments, a polynucleotide such as mRNA present in the cells hybridizes 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.

[0073] Optionally, the cells on the surface, such as a tissue section, are removed from the surface after permeabilization. In one embodiment, the cells are removed by enzymatic degradation. Removal can be accomplished by, for instance, degradation with proteinase K.

[0074] Capture of analytes and labeling of unoccupied capture probesIP-2924-PCT / 531.2924WO01

[0075] During or following permeabilization, the surface in contact with the permeabilized cells is exposed to conditions suitable for promoting capture of analytes by surface-immobilized capture probes (FIG. 1, block 30). When capture probes include a moiety that will bind to a non-polynucleotide analyte, some of the capture probes are typically occupied by target non-polynucleotide analytes. For instance, when a capture domain includes an antibody, a subset of the total capture probes on the surface will be occupied by proteins derived from the cells, e.g., cells present in a tissue, that have an epitope to which the antibody specifically binds. When capture probes include a polynucleotide, some of the capture probes are typically occupied by a target polynucleotide analytes. For instance, when a capture domain includes a poly-T nucleotide sequence, a subset of the total capture probes on the surface will be occupied by mRNA molecules derived from the cells.

[0076] Following the interaction between a capture probe and an analyte and the optional removal of cells, such as a tissue section, the surface can be contacted with a secondary analyte that will bind to unoccupied capture probes (FIG. 1, block 40). As used herein, a “secondary analyte” is a molecule that will not interact with a capture probe occupied by the target analyte, but will interact with a capture probe that is not occupied by the target analyte. Examples of secondary analytes useful with non-polynucleotide analytes include, but are not limited to, amino acids (e.g., a protein such as an antibody) and an aptamer. Examples of secondary analytes useful with polynucleotide analytes include, but are not limited to, DNA, RNA, and hybrids of DNA and RNA. The skilled person will appreciate that the binding affinity of the secondary analyte for the capture probe should be less that the binding affinity of the non-polynucleotide analyte for the capture probe. The secondary analyte can include a label. In one embodiment, in those embodiments where a non- polynucleotide analyte is being analyzed, the secondary analyte can include a label.

[0077] The methods described herein provide a universal approach that is easily applied to spatial workflows for the analysis of polynucleotide analytes, non-polynucleotide analytes, or both. The secondary analyte can be considered a universal analyte because the signal is generated by exploiting unoccupied capture probes.

[0078] Measuring label associated with a surfaceIP-2924-PCT / 531.2924WO01

[0079] After addition of the secondary analyte, the amount of label present on a surface is determined (FIG. 1, block 50). It is this step that allows identification of suitable permeabilization conditions for cells on the surface, such as cells present in a tissue.Suitable permeabilization conditions result in release of analytes from cells to allow the analytes to diffuse and interact with capture probes. However, too much permeabilization can result in increased diffusion and reduced interaction between analytes and capture probes. Different permeabilization conditions can be evaluated by identifying the amount of label associated with a surface to which cells had been applied. Because only unoccupied capture probes are labeled, the most desirable permeabilization conditions are those resulting in the least amount of label associated with the surface. Identified permeabilization conditions for a specific type of cells or tissue can be used in subsequent experiments with that particular type of cell or tissue.

[0080] 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). This permits identification of specific areas on the surface where cells or a tissue had been applied, and may be useful when spatial sequencing libraries of a specific part of a tissue are desired. When imaging is used, the presence of label indicates a lack of analyte from the cells or tissue, thus an inverse image of the presence of analytes will be present (see Example 1 and FIG. 6).

[0081] In another embodiment, the total amount of label present on a surface to which cells or a tissue had been applied can be determined. In this aspect, the capture probes can include a cleavage site that allows removal of all capture probes associated with a surface. After removal, the resulting mixture of total label can be determined by, for instance, a plate reader or slide scanner. Using this method, the desired permeabilization conditions are those resulting in the least amount of label associated with the surface.

[0082] FIG. 2 shows a general block diagram of an illustrative method for determining permeabilization conditions for non-polynucleotide analytes. The method includes providing a surface with a plurality of attached capture probes (FIG. 2, block 10). As described herein, the capture probe is configured to interact with a non-polynucleotideIP-2924-PCT / 531.2924WO01analyte, and thus can include a capture domain that includes a moiety that will bind to a non-polynucleotide analyte. Examples of moieties include, but are not limited to, a polynucleotide (e.g., an aptamer), amino acids (e.g., a protein) and ligands (e.g., a drug, a cofactor, or a hormone). An illustration of a grafted surface that is useful in this method is shown in FIG. 3A, which illustrates an embodiment of a grafted surface 30 having one population of attached capture probe 32 that is an antibody. In order to evaluate different permeabilization conditions, multiple grafted surfaces are typically used, with each surface having, for instance, an applied tissue from the same source, such as consecutive serial sections from the same tissue. Permeabilization results in release of analytes from the tissue and binding to capture probes on the surface (FIG. 2, block 20). FIG. 3B illustrates the grafted surface 30 with analyte 36 bound to capture probe 32, and unoccupied capture probes. Optionally, the applied cells, such as a tissue, can be removed (FIG. 2, block 30)

[0083] Following treatment of the grafted surfaces to differing permeabilization conditions, the surfaces can be washed to remove unbound analyte and a secondary analyte added to the surfaces under conditions suitable for interaction with unoccupied capture probes and without disrupting the bound analytes (FIG. 2, block 40). As described herein, the secondary analyte can include label or include a molecule that allows attachment of a label.FIG. 3C illustrates the grafted surface 30 with analyte 34 bound to capture probe 32 and secondary analyte 36 bound to capture probe 32. The secondary analyte associated with the capture probe on the surface can be determined as described herein (FIG. 2, block 50).

[0084] FIG. 4 shows a general block diagram of an illustrative method for determining permeabilization conditions for polynucleotide analytes. The method includes providing a surface with a plurality of attached capture probes (FIG. 4, block 10). As described herein, the capture probe is configured to interact with a polynucleotide analyte, and thus typically includes a polynucleotide capture domain that is complementary to a target polynucleotide analyte present in cells or a tissue. An illustration of a grafted surface that is useful in this method is shown in FIG. 5A, which illustrates a grafted surface 30 having one population of capture probe 51 attached by the 5' end to the surface. Each capture probe 51 includes an anchor domain 52 and a capture domain 53. In order to evaluate different permeabilization conditions, multiple grafted surfaces are typically used, with each surfaceIP-2924-PCT / 531.2924WO01having an applied tissue or cells from the same source, such as consecutive serial sections from the same tissue.

[0085] Permeabilization results in release of analytes (e.g., mRNA) from the cells, such as a tissue, and hybridization with capture probes on the surface (FIG. 4, block 20). An illustration of mRNA hybridizing to a capture probe is depicted in FIG. 5B. mRNAs 54 and 55 include a poly-A tail 56 that hybridizes to a poly-T region of the capture domain 53 of capture oligo 51 (FIG. 5B). Optionally, the cells, such as a tissue section, can be removed from the surface after the hybridization (FIG. 4, block 30).

[0086] The method includes use of a polymerase to produce a first complementary sequence (FIG.4, block 40). The polymerase extends the 3’ end of the capture probe using the hybridized polynucleotide analyte as a template. In one embodiment, when the analyte is RNA the polymerase can be a reverse transcriptase.

[0087] The result of the extension is a surface-attached first complementary sequence that includes the capture probe and a DNA sequence that is complementary (cDNA) to the hybridized polynucleotide analyte. When the hybridized analyte is mRNA and the capture probe’s capture domain includes a poly-T region or disrupted homopolymer, the first complementary sequence includes the complement of the full length of the mRNA. Tn other embodiments, for instance when the capture domain includes a randomer domain or a target-specific domain, the first complementary sequence can include the complement of a portion of the mRNA sequence. The hybridized polynucleotide analytes can be removed after the extension (FIG. 4, block 50). Alternatively, the hybridized polynucleotide analytes can be left in place, as their presence may increase the efficiency of labeling unoccupied capture probes.

[0088] In one embodiment, labeling of unoccupied capture probes can be accomplished by use of a terminal deoxynucleotidyl transferase (TdT), an enzyme that catalyzes the addition of dNTPs to the 3' hydroxyl terminus of DNA molecules. When a TdT is to be used, the extension reaction typically includes a terminator to inhibit the extension by a polymerase, resulting in a cDNA that terminates in a nucleotide that cannot be a substrate of a TdT. Examples of terminators include, but are not limited to, dideoxynucleotide (ddNTP) and 3’IP-2924-PCT / 531.2924WO01inverted dT. Following extension in the presence of a terminator and optional removal of the hybridized polynucleotide analytes, the surface can be exposed to a TdT and a labeled dNTP. The 3’ end of occupied capture probes include a terminator, thus the only substrate available for the TdT is the 3’ end of the unoccupied capture probes. The concentration of TdT and labeled dNTP can be altered to add a number of labeled dNTP suitable to obtain a desired level of signal.

[0089] An illustration of the use of a TdT is shown in FIG. 5C. The extension products 54’ and 55’ are terminated prematurely due to the presence of a terminator during the extension reaction. After optional removal of the hybridized polynucleotide analytes 54 and 55 (FIG.5D), TdT can be used to add labeled dNTPs 57 (with label depicted as stars) to the 3’ end of the unoccupied capture domain 53 of capture oligo 51. The amount of label associated with the surface can be determined as described herein (FIG. 4, block 80).

[0090] In those embodiments where a terminator is not included during an extension by a polymerase that uses hybridized polynucleotide analytes as template, extension typically results in longer cDNAs that are complementary to the entire template. An illustration of an extension 54' and 55' of a capture probe by a polymerase in the absence of a terminator is depicted in FIG. 5E, and an example of the result of an extension is depicted in FIG. 5F.The capture domain 53 of capture probe 51 has been extended to result in first complementary sequences 57 and 58. First complementary sequence 57 includes the complement of mRNA 54, the capture oligo 51, and is attached to surface 30.

[0091] In some embodiments, the double stranded structure resulting from extension of the 3’ end of capture probes hybridized to polynucleotide analytes can be selectively removed by exonuclease treatment before a secondary analyte is added. Removal of the double stranded portion can result in reducing any potential background during the subsequent labeling. Useful exonucleases are those that selectively degrade double stranded DNA and / or DNA-RNA hybrids and have minimal activity on single stranded DNA. Suitable exonucleases include, but are not limited to, Duplex DNAse (NEB, MA, USA) and T7 Exonuclease.IP-2924-PCT / 531.2924WO01

[0092] Following the production of the first complementary sequences, the surface is contacted with a secondary analyte that will bind to unoccupied capture probes (FIG. 4, block 60). An illustration of a secondary analyte hybridized to unoccupied capture probes is depicted in FIG. 5G. Secondary analyte 60 includes a poly-A tail 61 that hybridizes to a poly-T region of the capture domain 53 of capture oligo 51.

[0093] After hybridization of the secondary analyte to the unoccupied capture probes, those capture probes with a hybridized secondary analyte are labeled (FIG. 4, block 70). In one embodiment, a polymerase is used to extend the 3’ end of the capture probe hybridized to the secondary analyte, where the polymerase uses the hybridized secondary analyte as the template. A labeled dNTP can be included in the extension reaction, and the result of the extension is a labeled extension product.

[0094] An illustration of an extension 60' of a capture probe by a polymerase using the secondary analyte as template is depicted in FIG. 5H, and an example of the result of an extension is depicted in FIG. 51. The capture domain 53 of capture probe 51 has been extended to result in first complementary sequences 62. When the extension occurs in the presence of one or more labeled dNTPs, the extension reaction incorporates the labeled dNTPs (depicted as stars) and results converting unoccupied capture probes to labeled capture probes. An extension reagent useful in an extension reaction typically includes nucleotide triphosphates (NTPs). The NTPs include dATP, dTTP, dCTP, dGTP, or a subset of these dNTPs, e.g., just one dNTP, two of the dNTPs, or three dNTPs. In one embodiment, one or more of the dNTPs is labeled.

[0095] In another embodiment, capture probes with a hybridized secondary analyte can be labeled by using the hybridized secondary analyte as a splint polynucleotide. As used herein a “splint polynucleotide” refers to a polynucleotide that includes a nucleotide sequence complementary to a capture domain of a capture probe and another nucleotide sequence complementary to a labeled polynucleotide. After hybridization of a polynucleotide secondary analyte, a labeled polynucleotide can be added. The labeled polynucleotide hybridizes to the complementary sequence of the secondary analyte to form a splintedIP-2924-PCT / 531.2924WO01complex, thereby resulting in the capture domain of the capture probe and the labeled polynucleotide into ligatable proximity. This structure can then be ligated.

[0096] For instance, after a secondary analyte 60 is hybridized to unoccupied capture probes 51 as depicted in FIG. 5G, a labeled polynucleotide can be added. As shown in FIG. 5J, the secondary analyte 60 acts as a splint polynucleotide, and addition of the labeled polynucleotide 64 (with label depicted as stars) results in a splinted complex that includes the secondary analyte 60, the labeled polynucleotide 64, and the capture probe 51. As shown in FIG. 5K, after ligation the surface includes two types of attached polynucleotides, one derived from the polynucleotide analyte (polynucleotides 57 and 58) and one derived from the secondary analyte (polynucleotide 65). While labeled polynucleotide 64 is essentially equivalent in length to the complementary sequence of the secondary analyte 64, the length of the labeled polynucleotide can be shorter, longer or equivalent to the length of the complementary sequence of the secondary analyte.Likewise, the amount of label associated with the labeled polynucleotide (e.g., the labeled polynucleotide 62 in FIG. 51 and the labeled polynucleotide 65 in FIG. 5K) also vary from one labeled molecule (e.g., a labeled dNTP) to multiple labeled molecules. The skilled person will appreciate that these variables can be used to tune the label present so that the amount of signal present is optimized for sensitivity.

[0097] The label present in the polynucleotide added to the unoccupied capture probes (e.g., the first complementary sequences 62 of FIG. 51 or the attached polynucleotides 65 of FIG.5K) can be determined as described herein (FIG. 4, block 80). Using a polynucleotide second analyte to determine permeabilization conditions provides improved signal linearity than current methods. In standard approaches the signal from label incorporated during synthesis of cDNA strand is random because of length and amount of incorporated label, making normalization challenging. In the methods described herein, the nature of the polynucleotide second analyte, the length of the cDNA and the amount of incorporated label, are known. This results in a linear correlation between signal and number of captured strands, and therefore improved normalization.IP-2924-PCT / 531.2924WO01

[0098] The methods described herein provide an improvement in enzyme compatibility. In current spatial workflows for determining permeabilization for polynucleotide analytes, label is incorporated during cDNA synthesis, which limits the reverse transcriptase to one compatible with the conditions and label used. In the methods described herein, reverse transcription is performed using standard conditions and the labeling after addition of a second analyte can be performed using any combination of enzymes, such as RNA- dependent polymerase, DNA-dependent polymerase, ligase, terminal deoxynucleotidyl transferase (TdT), and the like.

[0099] Compositions

[0100] 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. 3C and 5E-5H. Another example of a composition includes an extension reagent that is useful in producing the compositions shown in FIGs. 5E-5H. Such extension reagents are missing one dNTP, e.g., they include one dNTP, two dNTPs, or three dNTPS, and one of the dNTPs is labeled.

[0101] Kits

[0102] 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 grafted polynucleotide capture probes, grafted non-polynucleotide capture probes, or cografted capture probes that include one or more polynucleotide capture probes and / or non- polynucleotide capture probes. 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.

[0103] 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 producingIP-2924-PCT / 531.2924WO01sequencing libraries. In addition, the packaging material contains instructions indicating how the materials within the kit are employed to practice reaction in different applications, including spatial workflows such as spatial transcriptomics. 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.

[0104] 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.

[0105] As used herein, the terms “polynucleotide,” “nucleic acid,” “nucleotide sequence,” and “oligonucleotide” 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 one species 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 moieties 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. UsefulIP-2924-PCT / 531.2924WO01non-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. In the context of analytes from cells on a surface, such as a tissue, “target polynucleotide” and “target polynucleotide analyte” refers to the subset of polynucleotide(s) from within a starting population of polynucleotides.

[0106] 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-dependent 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 or sequencing 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.

[0107] As used herein, “label” refers to a detectable marker. 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). A detectable marker can be part of a secondary analyte (e.g., aIP-2924-PCT / 531.2924WO01labeled dNTP or amino acid that is part of an aptamer or protein, respectively).Alternatively, a secondary analyte can include a molecule that allows attachment of a label to the secondary analyte. For instance, a secondary analyte can include a biotin molecule, and a label can include a streptavidin or avidin molecule. Examples of detectable markers include, but are not limited to, fluorescent dyes, radioactive isotopes, enzyme tags, chemiluminescent compounds, and bioluminescent compounds. In those methods described herein that evaluate non-polynucleotide analytes, a secondary analyte (e.g., a protein or ligand) can include a label or a molecule that allows attachment of a label to the secondary analyte. In those methods described herein that evaluate polynucleotide analytes, a secondary analyte (e.g., a polynucleotide such as mRNA) can be used as a template for synthesis of a complementary strand, and one or more of the dNTPs used by a polymerase in the extension can include a label or a molecule that allows attachment of a label to the secondary analyte.

[0108] 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" of “handle” in a subsequent step to hybridize 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 complementary to 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.

[0109] 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,IP-2924-PCT / 531.2924WO01organs, tissues, and / or biologically active chemical compound(s), such as analogs or mimetics thereof. An example of an analog or mimetic of an organ or tissue includes, but is not limited to, an organoid.

[0110] As used herein, the terms "organism," "subject," are used interchangeably and refer to, for instance, a microbe (e.g., prokaryotic or eukaryotic), an animal, or a plant. An example of an animal is a mammal, such as a human.

[0111] 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, an organ 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, subcutaneousIP-2924-PCT / 531.2924WO01tissue 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.

[0112] As used herein, the term "each," when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection unless the context clearly dictates otherwise.

[0113] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0114] 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."

[0115] 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.

[0116] 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."

[0117] 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, andIP-2924-PCT / 531.2924WO01limited 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.

[0118] 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.

[0119] As used herein, "providing" in the context of, for instance, a composition, an article, cells, or a tissue, means making the composition, the article, or the tissue, purchasing the composition, the article, the cells, or the tissue, or otherwise obtaining the composition, the article, the cells, or the tissue.

[0120] 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.

[0121] 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 thatIP-2924-PCT / 531.2924WO01range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] Exemplary Aspects

[0126] Aspect 1. A surface comprising capture probes attached to the surface at the 5’ ends, wherein members of a first population of the attached capture probes comprise a 3’ end that is covalently attached to a first complementary strand comprising a nucleotide sequence complementary to one of a plurality of polynucleotides, wherein the first complementary strands of the first population of the attached capture probes do not comprise a label, wherein members of a second population of the attached capture probes comprise a 3’ end that is covalently attached to a second complementary strand that comprises at least one label, and wherein the second complementary strand of each member of the second population of the attached capture probes is identical.

[0127] Aspect 2. The surface of any of aspects 1 or 3-18, wherein the plurality of polynucleotides comprises genomic DNA fragments or RNA.IP-2924-PCT / 531.2924WO01

[0128] Aspect 3. The surface of any of aspects 2-18, wherein the RNA comprises RNA involved in protein synthesis, RNA involved in regulation, or a combination thereof.

[0129] Aspect 4. The surface of any of aspects 3-18, wherein the RNA involved in protein synthesis comprises mRNA.

[0130] Aspect 5. The surface of any of aspects 3-18, wherein the RNA involved in regulation comprises miRNA, siRNA, or a combination thereof.

[0131] Aspect 6. The surface of any of aspects 1-5 or 7-18, wherein the label comprises a labeled dNTP.

[0132] Aspect 7. The surface of any of aspects 1- 6 or 8-18, wherein the capture probes comprise a barcode, a spatial barcode, or both a barcode and a spatial barcode.

[0133] Aspect 8. The surface of any of aspects 1-7 or 9-18, wherein the surface is 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 plate.

[0134] Aspect 9. The surface of any of aspects 1-8 or 10-18, wherein each capture probe of the first population of capture probes comprises, from 5’ to 3’, an anchor domain, a capture domain, and the first complementary strand.

[0135] Aspect 10. The surface of any of aspects 1-9 or 11-18, wherein each capture probe of the second population of capture probes comprises, from 5’ to 3’, the anchor domain, the capture domain, and the second complementary strand.

[0136] Aspect 11. The surface of any of aspects 1-10 or 12-18, wherein the anchor domain comprises a universal sequencer and a spatial barcode.

[0137] Aspect 12. A surface comprising a plurality of capture probes attached to the surface, wherein each of a first population of the plurality of attached capture probes comprise a moiety bound to a non-polynucleotide analyte, wherein the attached capture probes comprising a moiety bound to a non-polynucleotide analyte do not comprise a label, wherein each of a second population of the plurality of attached capture probes comprise aIP-2924-PCT / 531.2924WO01secondary analyte, wherein the secondary analyte comprises at least one label, and wherein the secondary analyte of each member of the second population of the attached capture probes is identical.

[0138] Aspect 13. The surface of any of aspects 1-12 or 14-18, wherein the moiety comprises amino acids or a ligand.

[0139] Aspect 14. The surface of any of aspects 1-13 or 15-18, wherein the amino acids comprise an antibody.

[0140] Aspect 15. The surface of any of aspects 1-14 or 16-18, wherein the ligand comprises a drug, a cofactor, or a hormone.

[0141] Aspect 16. The surface of any of aspects 1-15 or 17-18, wherein the non-polynucleotide analyte comprises a carbohydrate, a lipid, a protein, a glycoprotein, a lipoprotein, or a combination thereof.

[0142] Aspect 17. The surface of any of aspects 1-16, wherein the capture probes comprise a barcode, a spatial barcode, or both a barcode and a spatial barcode.

[0143] Aspect 18. The surface of any of aspects 1-17, wherein the surface is 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 plate.

[0144] Aspect 19. A method for identifying permeabilization conditions, comprising:providing a surface comprising a plurality of attached capture probes and cells, for instance a tissue, wherein the cells comprise analytes, permeabilizing the cells to release the analytes under conditions suitable for the analytes to bind to the capture probes, thereby converting the plurality of attached capture probes to a first population of occupied capture probes and a second population of unoccupied capture probes; labeling the unoccupied capture probes; and determining the amount of the labeled associated with the surface.

[0145] Aspect 20. The method of any of aspects 19 or 21-46, wherein the analytes comprise polynucleotides.IP-2924-PCT / 531.2924WO01

[0146] Aspect 21. The method of any of aspects 19-20 or 22-46, wherein the polynucleotides comprise genomic DNA fragments or RNA.

[0147] Aspect 22. The method of any of aspects 19-21 or 23-46, wherein the RNA comprises RNA involved in protein synthesis, RNA involved in regulation, or a combination thereof.

[0148] Aspect 23. The method of any of aspects 19-22 or 24-46, wherein the RNA involved in protein synthesis comprises mRNA.

[0149] Aspect 24. The method of any of aspects 19-23 or 25-46, wherein the RNA involved in regulation comprises miRNA, siRNA, or a combination thereof.

[0150] Aspect 25. The method of any of aspects 19-24 or 26-46, wherein the label comprises a labeled dNTP.

[0151] Aspect 26. The method of any of aspects 19-25 or 27-46, wherein the capture probes comprise a moiety that can bind a non-polynucleotide analyte.

[0152] Aspect 27. The method of any of aspects 19-26 or 28-46, wherein the moiety comprises amino acids or a ligand.

[0153] Aspect 28. The method of any of aspects 19-27 or 29-46, wherein the amino acids comprise an antibody.

[0154] Aspect 29. The method of any of aspects 19-28 or 30-46, wherein the ligand comprises a drug, a cofactor, or a hormone.

[0155] Aspect 30. The method of any of aspects 19-29 or 31-46, wherein the non- polynucleotide analyte comprises a carbohydrate, a lipid, a protein, a glycoprotein, a lipoprotein, or a combination thereof.

[0156] Aspect 31. The method of any of aspects 19-30 or 32-46, wherein the capture probes comprise a barcode, a spatial barcode, or both a barcode and a spatial barcode.IP-2924-PCT / 531.2924WO01

[0157] Aspect 32. The method of any of aspects 19-31 or 33-46, wherein the surface is 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 plate.

[0158] Aspect 33. A method for identifying permeabilization conditions, comprising:providing a surface comprising attached capture probes and cells, such as a tissue, wherein the cells comprise a first analyte, wherein the first analyte comprises polynucleotides, wherein the capture probes are attached at the 5’ end, wherein the capture probes comprise a 3’ capture domain; permeabilizing the cells to release the polynucleotides; wherein a region of each of the polynucleotides hybridizes to the 3’ capture domain of a capture probe, thereby converting a plurality of capture probes to a first population of occupied capture probes and a second population of unoccupied capture probes; extending the 3’ end of the first population of capture probes using the hybridized polynucleotides as template to produce first complementary strands attached to the 3’ end of each first population capture probe, hybridizing a polynucleotide second analyte to the 3’ capture domain of the second population of capture probes, wherein the polynucleotide second analyte comprises a first region that hybridizes to the 3’ capture domain of the second population of capture probes, and a second region at the 5’ end that does not hybridize to the 3’ capture domain of the second population of unoccupied capture probes; using the hybridized polynucleotide second analyte to attach a label to the 3’ end of each second population capture probe; and determining the amount of the labeled associated with the surface.

[0159] Aspect 34. The method of any of aspects 19-33 or 35-46, wherein the polynucleotides hybridized to the 3’ capture domain of the capture probe comprises RNA involved in protein synthesis, RNA involved in regulation, or a combination thereof.

[0160] Aspect 35. The method of any of aspects 19-34 or 36-46, wherein the RNA involved in protein synthesis comprises mRNA.

[0161] Aspect 36. The method of any of aspects 19-35 or 37-46, wherein the RNA involved in regulation comprises miRNA, siRNA, or a combination thereof.IP-2924-PCT / 531.2924WO01

[0162] Aspect 37. The method of any of aspects 19-36 or 38-46, wherein the extending the 3’ end of the first population of capture probes comprises reverse transcription.

[0163] Aspect 38. The method of any of aspects 19-37 or 39-46, wherein the polynucleotides hybridized to the 3’ capture domain of the capture probe comprises DNA.

[0164] Aspect 39. The method of any of aspects 19-38 or 40-46, wherein the using comprises extending the 3’ end of the second population of capture probes using the hybridized polynucleotide second analyte as template to produce second complementary strands attached to the 3’ end of each second population capture probe, wherein the extending comprises incorporating a label to result in the labeled polynucleotides attached to the surface.

[0165] Aspect 40. The method of any of aspects 19-39 or 41-46, wherein the extending the 3’ end of the second population of capture probes comprises a DNA-dependent polymerase.

[0166] Aspect 41. The method of any of aspects 19-40 or 42-46, wherein the label comprises one or more labeled dNTPs.

[0167] Aspect 42. The method of any of aspects 19-41 or 43-46, wherein the extending the 3’ end of the second population of capture probes comprises an extension reagent that has one dNTP, two dNTPs, or three dNTPs.

[0168] Aspect 43. The method of any of aspects 19-42 or 44-46, wherein the using comprises hybridizing a splint polynucleotide to the hybridized polynucleotide second analyte, wherein the hybridized splint polynucleotide is in ligatable proximity to the 3’ end of each second population capture probe, wherein the splint polynucleotide comprises a label; and

[0169] further ligating the hybridized splint polynucleotide and the 3’ end of each second population capture probe.

[0170] Aspect 44. The method of any of aspects 19-43 or 45-46, wherein the method further comprises applying the cells to the surface.IP-2924-PCT / 531.2924WO01

[0171] Aspect 45. The method of any of aspects 19-44 or 46, wherein the method further comprises removing the cells from the surface.

[0172] Aspect 46. The method of any of aspects 19-45, wherein the method further comprises applying the tissue to the surface.

[0173] EXAMPLES

[0174] 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.

[0175] Example 1

[0176] Demonstration of Assay Performance

[0177] Permeabilization experiments were performed using cryosections of mouse cerebellum to determine useful permeabilization times. Flowcells were grafted with a single capture probe having a poly-T nucleotide sequence at the 3’ end using a cBot as a fluidic device to deliver reagents. A cryostat was used to serially section the tissues and mount them onto the grafted flowcells.

[0178] The sections were fixed with methanol and stained with Hematoxylin and Eosin. The sections were then permeabilized over different times (see FIG. 6) and reverse transcription was performed. After reverse transcription, tissue digestion, RNA removal, an exogenous RNA (in this case mouse kidney total RNA) was added. mRNA molecules in the total RNA containing a poly-A nucleotide sequence at the 3 ’ end were captured by the unused probes. Extension of the 3’ ends of the capture probes was performed using a reverse transcriptase in the presence of Cy3-dCTP. After washing the surfaces were imaged with a fluorescence microscope.

[0179] For mouse cerebellum (FIG. 6), fluorescence images of the mouse cerebellum sections after 2 minutes, 5 minutes, 10 minutes, 15 minutes, 25 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 a decrease in intensity from 2 minutes toIP-2924-PCT / 531.2924WO015 minutes and a gradual increase in intensity from 5 minutes to 25 minutes. The lowest intensity was seen at 5 minutes. Analysis of these images indicates a useful permeabilization time for mouse cerebellum to be around 5 minutes.

[0180] Incorporation by Reference

[0181] 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, PIR, 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.

[0182] 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.

[0183] 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 examplesIP-2924-PCT / 531.2924WO01are 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.

[0184] 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

1. IP-2924-PCT / 531.2924WO01CLAIMS1. A surface comprising capture probes attached to the surface at the 5’ ends, wherein members of a first population of the attached capture probes comprise a 3’ end that is covalently attached to a first complementary strand comprising a nucleotide sequence complementary to one of a plurality of polynucleotides,wherein the first complementary strands of the first population of the attached capture probes do not comprise a label,wherein members of a second population of the attached capture probes comprise a 3’ end that is covalently attached to a second complementary strand that comprises at least one label, andwherein the second complementary strand of each member of the second population of the attached capture probes is identical.

2. The surface of claim 1, wherein the plurality of polynucleotides comprises genomic DNA fragments or RNA.

3. The surface of claim 2, wherein the RNA comprises RNA involved in protein synthesis, RNA involved in regulation, or a combination thereof.

4. The surface of claim 3, wherein the RNA involved in protein synthesis comprises mRNA.

5. The surface of claim 3, wherein the RNA involved in regulation comprises miRNA, siRNA, or a combination thereof.

6. The surface of claim 1, wherein the label comprises a labeled dNTP.

7. The surface of claim 1, wherein the capture probes comprise a barcode, a spatial barcode, or both a barcode and a spatial barcode.IP-2924-PCT / 531.2924WO018. The surface of claim 1, wherein the surface is 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 plate.

9. The surface of claim 1, wherein each capture probe of the first population of capture probes comprises, from 5’ to 3’, an anchor domain, a capture domain, and the first complementary strand.

10. The surface of claim 9, wherein each capture probe of the second population of capture probes comprises, from 5’ to 3’, the anchor domain, the capture domain, and the second complementary strand.

11. The surface of claim 10, wherein the anchor domain comprises a universal sequencer and a spatial barcode.

12. A surface comprising a plurality of capture probes attached to the surface, wherein each of a first population of the plurality of attached capture probes comprise a moiety bound to a non-polynucleotide analyte,wherein the attached capture probes comprising a moiety bound to a non-polynucleotide analyte do not comprise a label,wherein each of a second population of the plurality of attached capture probes comprise a secondary analyte, wherein the secondary analyte comprises at least one label, andwherein the secondary analyte of each member of the second population of the attached capture probes is identical.

13. The surface of claim 12, wherein the moiety comprises amino acids or a ligand.

14. The surface of claim 13, wherein the amino acids comprise an antibody.

15. The surface of claim 13, wherein the ligand comprises a drug, a cofactor, or a hormone.IP-2924-PCT / 531.2924WO0116. The surface of claim 12, wherein the non-polynucleotide analyte comprises a carbohydrate, a lipid, a protein, a glycoprotein, a lipoprotein, or a combination thereof.

17. The surface of claim 12, wherein the capture probes comprise a barcode, a spatial barcode, or both a barcode and a spatial barcode.

18. The surface of claim 12, wherein the surface is 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 plate.

19. A method for identifying permeabilization conditions, comprising:providing a surface comprising a plurality of attached capture probes and attached cells, optionally wherein the attached cells are present in a tissue,wherein the cells comprises analytes,permeabilizing the cells to release the analytes under conditions suitable for the analytes to bind to the capture probes, thereby converting the plurality of attached capture probes to a first population of occupied capture probes and a second population of unoccupied capture probes;labeling the unoccupied capture probes; anddetermining the amount of the labeled associated with the surface.

20. The method of claim 19, wherein the analytes comprise polynucleotides.

21. The method of claim 20, wherein the polynucleotides comprise genomic DNA fragments or RNA.

22. The method of claim 21, wherein the RNA comprises RNA involved in protein synthesis, RNA involved in regulation, or a combination thereof.IP-2924-PCT / 531.2924WO0123. The method of claim 22, wherein the RNA involved in protein synthesis comprises mRNA.

24. The method of claim 22, wherein the RNA involved in regulation comprises miRNA, siRNA, or a combination thereof.

25. The method of claim 20, wherein the label comprises a labeled dNTP.

26. The method of claim 19, wherein the capture probes comprise a moiety that can bind a non-polynucleotide analyte.

27. The method of claim 26, wherein the moiety comprises amino acids or a ligand.

28. The method of claim 27, wherein the amino acids comprise an antibody.

29. The method of claim 27, wherein the ligand comprises a drug, a cofactor, or a hormone.

30. The method of claim 26, wherein the non-polynucleotide analyte comprises a carbohydrate, a lipid, a protein, a glycoprotein, a lipoprotein, or a combination thereof.

31. The method of claim 19, wherein the capture probes comprise a barcode, a spatial barcode, or both a barcode and a spatial barcode.

32. The method of claim 19, wherein the surface is 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 plate.

33. A method for identifying permeabilization conditions, comprising:providing a surface comprising attached capture probes and attached cells, optionally wherein the attached cells are present in a tissue,IP-2924-PCT / 531.2924WO01wherein the cells comprise a first analyte, wherein the first analyte comprises polynucleotides,wherein the capture probes are attached at the 5’ end,wherein the capture probes comprise a 3’ capture domain;permeabilizing the cells to release the polynucleotides;wherein a region of each of the polynucleotides hybridizes to the 3’ capture domain of a capture probe, thereby converting a plurality of capture probes to a first population of occupied capture probes and a second population of unoccupied capture probes; extending the 3’ end of the first population of capture probes using the hybridized polynucleotides as template to produce first complementary strands attached to the 3’ end of each first population capture probe,hybridizing a polynucleotide second analyte to the 3’ capture domain of the second population of capture probes,wherein the polynucleotide second analyte comprises a first region that hybridizes to the 3’ capture domain of the second population of capture probes, and a second region at the 5’ end that does not hybridize to the 3’ capture domain of the second population of unoccupied capture probes;using the hybridized polynucleotide second analyte to attach a label to the 3’ end of each second population capture probe; anddetermining the amount of the labeled associated with the surface.

34. The method of claim 33, wherein the polynucleotides hybridized to the 3’ capture domain of the capture probe comprises RNA involved in protein synthesis, RNA involved in regulation, or a combination thereof.

35. The method of claim 34, wherein the RNA involved in protein synthesis comprises mRNA.

36. The method of claim 34, wherein the RNA involved in regulation comprises miRNA, siRNA, or a combination thereof.IP-2924-PCT / 531.2924WO0137. The method of any one of claims 34-36, wherein the extending the 3’ end of the first population of capture probes comprises reverse transcription.

38. The method of claim 33, wherein the polynucleotides hybridized to the 3’ capture domain of the capture probe comprises DNA.

39. The method of claim 33, wherein the using comprises extending the 3’ end of the second population of capture probes using the hybridized polynucleotide second analyte as template to produce second complementary strands attached to the 3’ end of each second population capture probe,wherein the extending comprises a incorporating a label to result in the labeled polynucleotides attached to the surface.

40. The method of claim 39, wherein the extending the 3’ end of the second population of capture probes comprises a DNA-dependent polymerase.

41. The method of claim 39, wherein the label comprises one or more labeled dNTPs.

42. The method of claim 39, wherein the extending the 3’ end of the second population of capture probes comprises an extension reagent that has one dNTP, two dNTPs, or three dNTPs.

43. The method of claim 33, wherein the using comprises hybridizing a splint polynucleotide to the hybridized polynucleotide second analyte, wherein the hybridized splint polynucleotide is in ligatable proximity to the 3’ end of each second population capture probe, wherein the splint polynucleotide comprises a label; andfurther ligating the hybridized splint polynucleotide and the 3’ end of each second population capture probe.

44. The method of claim 33, wherein the method further comprises applying the cells to the surface.IP-2924-PCT / 531.2924WO0145. The method of claim 33, wherein the method further comprises removing the cells from the surface.

46. The method of claim 33, wherein the method further comprises applying the tissue to the surface.

47. The method of claim 33, wherein the method further comprises removing the tissue from the surface.

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