WO2025097072A2

WO2025097072A2 - Methods and compositions for analyzing cells and their constituents

Info

Publication number: WO2025097072A2
Application number: PCT/US2024/054290
Authority: WO
Inventors: George HARTOULAROS; Lin PHAM; Alyssa WARD; Joshua MAST; Eric Chow; Chun Ye
Original assignee: Survey Genomics Inc; University of California Berkeley; University of California San Diego UCSD
Current assignee: Survey Genomics Inc; University of California Berkeley; University of California San Diego UCSD
Priority date: 2023-11-02
Filing date: 2024-11-01
Publication date: 2025-05-08
Anticipated expiration: 2026-05-02
Also published as: WO2025097072A3

Abstract

A method for labeling cells or a subcellular compartment thereof is provided. The method may comprise: providing an array comprising a plurality of features each comprising one or more nucleic acid barcode molecules that comprise: a non-nucleotidyl moiety that binds to cells or a subcellular compartment thereof and a spatial barcode sequence that alone or in combination with one or more other spatial barcode sequences in a feature identify the location of the feature; contacting a tissue sample with the array and allowing the nucleic acid barcode molecules to couple to the cells or a subcellular compartment thereof in the tissue sample; removing the tissue sample from the array; and disassociating the cells or subcellular compartment thereof in the tissue sample to produce a suspension comprising single cells or subcellular compartments that are labeled with one or more spatial barcode sequences. Kits for performing the method are also provided.

Description

METHODS AND COMPOSITIONS FOR ANALYZING CELLS AND THEIR CONSTITUENTS

CROSS-REFERENCING

[0001] This application claims the benefit of U.S. provisional application serial no. 63/595,412, filed on November 2, 2023, which application is incorporated by reference in its entirety.

BACKGROUND

[0002] Determining the location of cells and their constituents in a biological sample can provide understanding of the function of cells and cell types as well as the interactions and organization of cells.

SUMMARY

[0003] A method for labeling cells or a subcellular compartment thereof is provided. In some embodiments, the method may comprise: (a) providing an array comprising a plurality of features each comprising one or more nucleic acid barcode molecules that comprise: (i) a non-nucleotidyl moiety that binds to cells or a subcellular compartment thereof, and (ii) a spatial barcode sequence that alone or in combination with one or more other spatial barcode sequences in a feature identifies a location of the feature on the array; (b) contacting a tissue sample with the array and, while the tissue sample is contacted with the array, allowing the nucleic acid barcode molecules to couple to the cells or a subcellular compartment thereof in the tissue sample; (c) removing the tissue sample from the array; and (d) disassociating the cells or subcellular compartment thereof in the tissue sample after it has been removed from the array to produce a suspension comprising single cells or subcellular’ compartments that are labeled with one or more spatial barcode sequences that identify a location of a feature on the array. Kits for performing the method are also provided.

[0004] In an aspect, the present disclosure provides a method for identifying a position of a cell, cellular compartment, or secreted protein of a tissue sample, comprising: (a) providing an array comprising a plurality of features, wherein a feature of the plurality of features comprises a nucleic acid barcode molecule comprising a barcode sequence, wherein the barcode sequence identifies a location of the feature on the array; (b) contacting the tissue sample with the array, wherein, the cell, cellular compartment, or secreted protein of the tissue sample is positioned at the feature on the array upon the contacting; (c) coupling the nucleic acid barcode molecule to the cell, cellular compartment, or secreted protein at the feature; (d) identifying the barcode sequence of the nucleic acid barcode molecule, thereby identifying the location of the feature of the array; and (e) using the location of the feature of the array to identify the position of the cell, cellular compailment, or secreted protein in the tissue sample.

[0005] In some embodiments, the feature comprising a nucleic acid barcode molecule further comprises a binding moiety. In some embodiments, the binding moiety comprises a lipid. In some embodiments, the binding moiety comprises a protein. In some embodiments, the protein comprises an antibody or a fragment of an antibody. In some embodiments, the binding moiety comprises a nucleic acid. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is DNA.

[0006] In some embodiments, (c) comprises coupling the binding moiety to the cell, cellular compartment, or secreted protein. In some embodiments, (c) comprises coupling the binding moiety to a surface protein on the cell, cellular compartment, or secreted protein. In some embodiments, the binding moiety is coupled to a nucleic acid capture sequence, and wherein the nucleic acid barcode molecule is configured to hybridize with the nucleic acid capture sequence. In some embodiments, the nucleic acid barcode molecule further comprises a poly-adenosine sequence.

[0007] In some embodiments, the array is a microwell array and the feature is a microwell. In some embodiments, the array comprises a planar surface. In some embodiments, during or after (b), the tissue sample is sandwiched between the array and another surface. In some embodiments, the other surface is another array. In some embodiments, the nucleic acid barcode molecule is immobilized at the feature. In some embodiments, the nucleic acid barcode molecule is reversibly immobilized to the feature. In some embodiments, (c) comprises releasing the nucleic acid barcode molecule from the feature. [0008] In some embodiments, the nucleic acid barcode molecule is reversibly immobilized to the feature through a photocleavable linkage. In some embodiments, the nucleic acid barcode molecule is reversibly immobilized to the feature through a matrix. In some embodiments, the matrix comprises a hydrogel.

[0009] In some embodiments, the method comprises in (a), the nucleic acid barcode molecule is in dry form at the feature. In some embodiments, the method further comprises, following (a), suspending the nucleic acid barcode molecule in fluid from the tissue sample. In some embodiments, the feature comprises preserved material. In some embodiments, the preserved material comprises frozen material.

[0010] In some embodiments, the method further comprises, following (c), enriching for the cell, cellular compailment, or secreted protein coupled to the nucleic acid barcode molecule from a plurality of cells or cellular compartments. In some embodiments, the enriching for a cell, cellular compartment, or secreted protein coupled to a nucleic acid barcode molecule comprises flow cytometry. In some embodiments, the feature comprises a fluorescent tag. In some embodiments, the enriching for the cell or cellular feature coupled to the nucleic acid barcode molecule comprises affinity-based enrichment. In some embodiments, the affinity-based enrichment comprises use of streptavidin, avidin, or biotin.

[0011] In some embodiments, the method further comprises, during or after (d), identifying a barcode sequence of a nucleic acid barcode molecule in a single cell assay. In some embodiments, the single cell assay comprises nucleic acid sequencing. In some embodiments, the single cell assay comprises flow cytometry. In some embodiments, the single cell assay comprises mass cytometry.

[0012] In some embodiments, the method further comprises, during or after (d), determining the location of the cell, cellular compartment, or secreted protein by determining a concentration of the nucleic acid barcode. In some embodiments, the tissue sample comprises a tissue section, a cell monolayer, an organoid, a fixed cell, or any combination thereof. In some embodiments, the method further comprises, during or after (b), dissociating the tissue into single cells comprising the cell, cellular compartment, or secreted protein. In some embodiments, the method further comprises, during or after (c), dissociating the tissue into single cells comprising the cell, cellular compartment, or secreted protein.

[0013] In some embodiments, the method further comprises, during or after (e), using the location of the cell, cellular compartment, or secreted protein to determine a position of an analyte of the cell, cellular compartment, or secreted protein in the tissue.

[0014] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “figure” and “FIG.” herein), of which:

[0017] FIG. 1 shows an example scheme for features on an array.

[0018] FIGs. 2A-2C show example schemes for an array configuration. FIG. 2A shows an example scheme for an array and tissue configuration. FIG. 2B shows an example scheme for a configuration of multiple arrays (or a cover and an array) and a tissue. FIG. 2C shows an example scheme for a configuration of multiple arrays (or a cover and an array) and a tissue.

[0019] FIG. 3 shows an example scheme for a feature comprising an immobilized barcode molecule.

[0020] FIGs. 4A-4C show example schemes for binding moieties and corresponding barcodes. FIG. 4A shows an example scheme for barcoded cells in a well of an array. FIG. 4B shows an example scheme for barcoded binding moieties. FIG. 4C shows an example scheme for barcoded binding moieties.

[0021] FIGs. 5A-5C show example schemes for labeling cells. FIG. 5A shows an example scheme for barcoded binding moieties and potential labeling configurations. FIG. 5B shows an example scheme for reverse transcription of the spatial barcode and droplet barcode. FIG. 5C shows an example scheme for reverse transcription of the gene transcripts and droplet barcode.

[0022] FIGs. 6A-6D show example schemes for labeling cells. FIG. 6A shows an example scheme for barcoded binding moieties and potential labeling configurations. FIG. 6B shows an example scheme for reverse transcription of the spatial barcode and droplet barcode. FIG. 6C shows an example scheme for reverse transcription of the gene transcripts and droplet barcode. FIG. 6D shows an example scheme for reverse transcription of the antibody barcode and droplet barcode.

[0023] FIG. 7 shows an example scheme for barcoded binding moieties and potential labeling configurations.

[0024] FIG. 8 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0025] FIG. 9 illustrates how the present method unifies single-cell and spatial multi-omics profiling (e.g., RNA + Protein), with integrated sequencing read-out for fast analysis.

[0026] FIG. 10 illustrates an exemplary workflow of the present method.

[0027] FIG. 11 illustrates an example of how cells can be labeled with spatial barcodes using the present method.

[0028] FIG. 12 illustrates how labeling can occur. In this example, two fluorescent reagents are dispensed into the high density microarray (HDMA) in a checkerboard manner and a tissue slice is then placed on the array for spatial labeling. Tissues slices are examined for fluorescent signals using microscopy. A: Photo of a section of the HDMA after two fluorescent reagents are dispensed into it in a checkboard manner. B: Control experiment: PBS was dispensed into the HDMA without any fluorescent reagents, no colors are observed on the tissue after the procedure. C: Label experiment: Two fluorescent reagents are dispensed into the HDMA in a checkerboard manner to label the tissue slice. Fluorescent signals are observed in the respective dye emission channel.

[0029] FIG. 13 shows that the label can persist through dissociation.

[0030] FIG. 14 shows how data can be processed to generate multiomic data along with spatial context.

DETAILED DESCRIPTION

[0031] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0032] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0033] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that scries of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0034] As used herein, the term “tissue sample” refers to a three-dimensional sample that comprises multiple cells (e.g., at least 50, at least 100, at least 500, at least 1,000, at least 5,000 or at least 10,000 cells) that are spatially associated with one another, e.g., via an extracellular matrix. Tissue sections, cell monolayers, and organoids are examples of a tissue sample because they contain multiple cells that are spatially associated with another. This term is intended to exclude suspensions of single cells. A tissue section may have a thickness of at least 1 micrometers (e.g., at thickness in the range of 1 um to 5 mm, 1 um to 1 mm, 1 um to 100 um, 5 um to 500 um, 10 um to 20 um, 20 um to 100 um, etc., although tissue sections that have a thickness outside of this range can be used in many circumstances).

[0035] As used herein, the term “dissociating” refers to a step in which cells or subcellular compartments in a tissue sample are separated from another to produce a suspension that contains single cells or subcellular compartments, where the single cells or subcellular compartments are not physically connected to each other. Single cell suspensions may be made by treating the tissue with one or more enzymes e.g., one or more proteolytic or collagenolytic enzymes such as trypsin or Accutase, or by treating the tissue with EDTA. See, e.g., Lai Scientific Reports 2022 12: 5713. Subcellular components such as the nucleus may be released by the addition of a permeabilization agent (e.g., a detergent such as Tween or NP40). A suspension that comprises single cells or subcellular compartments may also contain cell debris and clumps that include multiple cells or compartments. If necessary, these things can be eliminated prior to the next steps.

[0036] The terms “subcellular compartment” and “cellular compartment” refer to the nucleus as well as non-nuclear cellular compartments selected from the group consisting of cytoplasm, endoplasmic reticulum, Golgi apparatus, mitochondrion, endosome, lysosome, secretory vesicle, cilium, plasma membrane, vacuole, and peroxisome.

[0037] In the context of this disclosure, a “spatial barcode sequence” refers to the same thing as “a barcode sequence that identifies a location of a feature on an array”. A location of a feature on an array may be defined by x-y coordinates, for example. As noted below, a spatial barcode sequence can identify a location of the feature on the array on its own or in combination with one or more other spatial barcode sequences in a feature. In the former case, each feature will contain a spatial barcode sequence that is different in each feature. In the latter case, each feature will contain a combination of spatial barcode sequences, where the combination of sequences is different in each feature. In the latter case, each feature may contain up to five (e.g., two, three, four or five) or more than five spatial barcode sequences, where the combination of spatial barcode sequences in a feature uniquely identifies each feature from other features on the array. In one exemplary embodiment, if the features each contain two spatial barcode sequences, then each feature of an array of 100 features can be uniquely identified using only 20 spatial barcode sequences. In another example, if the features contain three spatial barcode sequences, then each feature of an array of 1000 features can be uniquely identified using only 30 spatial barcode sequences. For example, each feature of an array of 1000 features can be uniquely identified using 64 spatial barcodes.

[0038] The term “array” refers to an object that comprises a substrate (e.g., a microscope slide or a microwell plate) that contains discrete non-overlapping areas (which are referred to as “features” or “elements”) that contain defined reagents, as described below. As will be described in greater detail below, an array may comprise at least at least 100, at least 384, at least 1,000, at least 10,000, at least 50,000 or at least 100,000 features, where each feature contains a spatial barcode or combination of spatial barcodes (see above) that indicates a location of a feature on the array (e.g., by its x-y coordinates). A substrate may, for example, have the dimensions of a standard microscope slide or a coverslip and the features may collectively occupy an area up to 1 cm x 2 cm or more (typically more than 0.1 cm x 0.1 cm) of the substrate. The features themselves may have an average diameter in the range of 1 micrometer to 100 micrometers (e.g., 1 micrometer to 50 micrometers or 1 micrometer to 10 micrometers), although features having diameters outside of this range can be readily used.

[0039] The term “non-nucleotidyl” refers to a moiety that is not a nucleic acid. Oligo(dT), polyadenosine (poly(A)), random primers and oligonucleotide probes are excluded by this definition. Non-nucleotidyl moieties do not hybridize and cannot be extended by a polymerase. Proteins (e.g., antibodies) and lipids are examples of non-nucleotidyl moieties. In any embodiment, a binding moiety can be a non-nucleotidyl in any embodiment.

[0040] The term “binds to cells or a subcellular compartment thereof’ refers to binding events that occur anywhere in or on a cell, including the cell surface (by insertion into the plasma membrane or binding to cell surface proteins) and binding events that are within a cell, e.g., in or on a subcellular compartment, such as the nucleus etc.

[0041] The term “omics” refers to the analysis of (i.e., detecting the presence and/or abundance of) multiple analytes (e.g., genome alterations, nucleotide modifications, e.g., methyl C, chromatin structure, the transcriptome and/or the expression of multiple proteins) on a cell-by-cell basis in the same workflow. The term “omics” analysis is intended to include perturb-seq (see, e.g., Dixit et al Cell 2016 167: 1853-1866) applications in which the omics nucleic acids and perturbation barcodes are both captured in the omics library.

[0042] The term “universal processing sequence” refers to a sequence of at least 12 contiguous nucleotides (e.g., at least 15 or at least 18 nucleotides) that may be used as a primer (e.g., may be used as a reverse transcription primer, such as oligo(dT), or a random primer), may provide a binding site for a primer (e.g., poly-adenosine (poly(A)), or a “PCR handle”), or may provide a binding site for a probe (e.g., for enrichment). This sequence provides compatibility with the components used in a downstream workflow and, since the downstream workflow may be implemented in a variety of different ways, the sequence may vary depending on how the downstream workflow is implemented. For example, this sequence have poly-adenosine, the sequence may have the sequence of a template switching oligonucleotide, a PCR primer binding site and/or a sequence that is compatible with oligonucleotide that is tethered to a bead (for sequence enrichment, etc.). In any embodiment, universal processing sequence may be a primer, a primer binding site or a binding site for probe, where the sequence is at least 12 nucleotides in length. The “universal processing sequence” may be referred to as the “barcode capture sequence” in certain parts of this disclosure.

[0043] The term “enriching” in the context of enriching for cells in a suspension, refers to a step that may be done by any suitable method, including flow cytometry or affinity capture. In some embodiments, a cell may be stained (e.g., with a viability stain) or labeled (e.g., with a labeled antibody) before selection. The terms “selecting” and “enriching for” have the same meaning. For example, a certain cell type (e.g., T cells or the like) may be selected from the suspension.

[0044] Recognized herein is a need for improved spatial biology methods and compositions. [0045] Spatial biology methods and compositions described herein can help elucidate the interactions and organization of cells and their constituents in the context of a tissue or other multicellular system. The methods and compositions disclosed herein provide spatial information at a high resolution and can be coupled to a range of downstream assays. Such downstream assays include single cell assays including those performed in droplets and microwells. Such compositions and methods can make use of spatial tags such as nucleic acid barcodes that can be read out to determine positional information of a cell and, thus, its constituents in a tissue or other type of biological sample.

[0046] A method for labeling cells or a subcellular compartment thereof is provided. In some embodiments, the method may comprise: (a) providing an array comprising a plurality of features each comprising one or more nucleic acid barcode molecules that comprise: (i) a non-nucleotidyl moiety that binds to cells or a subcellular compartment thereof, and (ii) a spatial barcode sequence that alone or in combination with one or more other spatial barcode sequences in a feature identifies a location of the feature on the array; (b) contacting the tissue sample with the array and, while the tissue sample is contacted with the array, allowing the nucleic acid barcode molecules to couple to the cells or a subcellular compartment thereof in the tissue sample; (c) removing the tissue sample from the array; and (d) disassociating the cells or subcellular compartment thereof in the tissue sample after it has been removed from the array to produce a suspension comprising single cells or subcellular compartments that are labeled with one or more spatial barcode sequences that identify a location of a feature on the array.

[0047] In some embodiments, the method may comprise: (e) making a single-cell sequencing library from the suspension a subset of cells thereof, or a subcellular compartment of either; (f) sequencing the single-cell sequencing library to produce sequence reads; (g) identifying one or more spatial barcode sequences in the sequence reads, and (h) mapping the location of a cell or subcellular compartment to a feature of the array using the one or more spatial barcode sequences. In these embodiments: the single-cell sequencing library may be an omics library that comprises: (i) polynucleotides that comprise an analyte-related nucleic acid linked to a cell-specific barcode; and (ii) polynucleotides that comprise a spatial barcode linked to a cell- specific barcode, the sequencing may produce omics data (which, as described below, may include data on the transcriptome, protein expression, chromatin structure, etc., on a cell-to-cell basis); and the method may further comprise: mapping the omics data for an individual cell or subcellular compartment to a feature of the array using the one or more spatial barcodes for the individual cell or subcellular compartment. In some embodiments, the method may comprise reconstructing an image of the tissue sample using the mapped omics data. In some embodiments, the method may be done on multiple sections of the same tissue. In these embodiments, a three-dimensional image of the tissue may be constructed using the data produced by the method. An exemplary workflow for how this embodiment of the method may be performed is illustrated in FIG. 10. Examples of types of omics data that can be generated using the present method are illustrated in FIG. 14.

[0048] In alternative embodiments, the spatial barcodes may be read in situ, by hybridizing labeled probes to the spatial barcodes. In some embodiments, this in situ-based approach may involve multiple rounds of probe binding, hybridization and imaging. See, e.g., Goltsev et al Cell. 2018 174:968-981.

[0049] In any embodiment, the non-nucleotidyl moiety may be a binding agent (e.g., an antibody) that recognizes a protein in or on a cell or subcellular compartment or a lipid that integrates into a membrane of the cell or subcellular compartment (e.g., a fatty acid such as lignoceric acid or palmitic acid and/or cholesterol; see, e.g., McGinnis et al Nature Methods 2019 16: 619-626). In embodiments wherein the non-nucleotidyl moiety binds to a subcellular compartment, the cells in the sample may be fixed and/or permeabilized prior to coupling.

[0050] In any embodiment, a nucleic acid barcode molecule may comprise: (i) a first oligonucleotide that comprises a capture sequence and the non-nucleotidyl moiety; and (ii) a second oligonucleotide comprising a sequence that is complementary to the capture sequence of the first oligonucleotide, and the spatial barcode, as illustrated in Figs. 4B and 4C. The second oligonucleotide may further comprise one or more universal processing sequences.

[0051] In an aspect, the present disclosure provides a method for identifying a position of a cell, subcellular compartment, or secreted protein of a tissue sample. The method can include: (a) providing an array comprising a plurality of features. A feature of the plurality of features can include a nucleic acid barcode molecule comprising a barcode sequence. The barcode sequence can identify a location of the feature on the array. The method can also include (b) contacting the tissue sample with the array. In some cases, the cell, subcellular compartment, or secreted protein of the tissue sample may be positioned at the feature on the array upon the contacting. In addition, the method can include (c) coupling the nucleic acid barcode molecule to the cell, subcellular compartment, or secreted protein at the feature. The method can also include (d) identifying the barcode sequence of the nucleic acid barcode molecule, thereby identifying the location of the feature of the array. Additionally, the method can include (e) using the location of the feature of the array to identify the position of the cell, cellular compartment, or secreted protein in the tissue sample.

[0052] In any embodiment, steps (b) and (c) of this method (the ‘contacting’ and ‘coupling’ steps in which the tissue sample is placed in contact with the array and the nucleic barcode molecule is coupled with the cell, subcellular compartment, or secreted protein) may be done without disassociating the cells, i.e., using an intact tissue that has not been treated with a disassociation agent (such as a proteolytic or collagenolytic enzyme, or EDTA). In this case, the cells in the tissue sample make direct contact with the nucleic acid barcode molecules. If the nucleic acid barcode molecules are in wells then, generally, the molecules will be in solution and, in some embodiments, a force may be applied to facilitate contact between the tissue sample and the nucleic acid barcode molecules. For example, the tissue sample may be pressed into the array, thereby deforming the tissue sample such that parts of the tissue sample that are over the entrances to the wells become deformed and are pushed into the wells. Alternatively (and as another example), the solution containing the nucleic acid barcode molecules can be forced into the tissue section by centrifugal force, suction, electrophoresis or by magnetism, for example. In some embodiments, e.g., if the array is planar or the wells are full close to the brim, no force need be applied. The tissue section may remain largely intact in this part of the method. However, some breakage or tearing may occur in handling or due to the pressure of being pushed into wells. In some embodiments, the nucleic acid barcode molecules may be in dry form on the array and become hydrated once they are in contact with the tissue sample. An example of how this step of the method may be performed is illustrated in

FIG. 11.

[0053] The feature comprising a nucleic acid barcode molecule may further comprise a binding moiety. The binding moiety may comprise a lipid, a protein, a polypeptide, a nucleic acid molecule, an aptamer, a small molecule, a ligand, or a combination thereof. In some cases, the binding moiety comprises one or more proteins, combinations of proteins, one or more lipids, combinations of lipids, or combinations of lipids and proteins. The binding moiety may comprise one or more proteins, one or more small molecules, or combinations of proteins and small molecules. The binding moiety may comprise one or more lipids, one or more small molecules, or combinations of lipids and small molecules.

[0054] In some cases, the binding moiety comprises a lipid. The binding moiety may comprise one or more fatty acids, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, sphingomyelins, cholesterols, glycolipids, phospholipids, phospholipases, glycerophospholipids, sphingolipids, sterols, or a combination thereof. In some embodiments, the one or more fatty acids may comprise lignoceric acid, palmitic acid, lauric acid, stearic acid, or a combination thereof. The lipid may bind to a component on a cell, cellular compartment, or secreted protein that is specific to a particular cell type. The lipid may bind to a component on a cell, cellular compartment, or secreted protein that is expressed on all or nearly all cell types within a given tissue.

[0055] A pool of lipid binding moieties can comprise multiple groups of lipid binding moieties, each of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of lipid binding moieties in the pool. In some examples, a pool of lipid binding moieties comprises multiple groups of lipid binding moieties, some of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of lipid binding moieties in the pool. A pool of lipid binding moieties, in some cases, comprises multiple groups of lipid binding moieties, each of which bind to the same or similar components on a cell, cellular compartment, or secreted protein as the other groups of lipid binding moieties in the pool.

[0056] In some embodiments, the binding moiety comprises a protein. The binding moiety may comprise a protein that binds to the surface of a cell, cellular compartment, or secreted protein. In some cases, the protein binding moiety may bind to one or more cell surface proteins. The protein binding moiety may bind to a component that is specific to a particular’ cell type. A component may comprise a cell surface protein, a transmembrane protein, a post-translational modification on a protein, a polypeptide, a lipid, a carbohydrate, or a combination thereof. The protein binding moiety may bind to a component that is specific to a particular cell, cellular compartment, or secreted protein. The protein binding moiety may bind to a component that is expressed on all or nearly all cell types within a given tissue. The protein binding moiety may bind to a component that is expressed on a cellular compartment in all or nearly all cell types within a given tissue.

[0057] A pool of protein binding moieties can comprise multiple groups of protein binding moieties, each of which binds to a different component on a cell, cellular compartment, or secreted protein than the other groups of protein binding moieties in the pool. In some cases, a pool of protein binding moieties can comprise multiple groups of protein binding moieties, some of which bind to a different component on a cell, cellular' compartment, or secreted protein than the other groups of protein binding moieties in the pool. Moreover, in some examples, a pool of protein binding moieties comprises multiple groups of protein binding moieties, each of which bind to the same or similar components on a cell, cellular compartment, or secreted protein as the other groups of protein binding moieties in the pool.

[0058] As described above, the non-nucleotidyl moiety binds to sites that are in or on cells. For example, if a proteinaceous binding moiety is used, the protein binding moiety may bind to a protein that is ubiquitously expressed in or on at least a subset of the cells being tested. For example, a combination of an anti-MHC-I antibody (which recognizes a surface protein present on all nucleated cells) and an anti-CD45 antibody (which recognizes a receptor protein tyrosine kinase present on all leukocytes) could be used. In some embodiments, the non-nucleotidyl binding agent may bind to the surface of the cell. For example, moieties (e.g., antibodies) that recognize cell surface markers, or lipid moieties could be used. Alternatively, moieties (e.g., antibodies) that recognize internal targets, or lipid moieties could be used. In these latter embodiments, the cells may be fixed and/or permeabilized (e.g., using a detergent) beforehand. [0059] The protein binding moiety may comprise an antibody, which term includes full length antibodies and fragments thereof that bind to the antigen, nanobodies, and all antibody variations, including scFvs, etc, an aptamer, or another protein binding scaffold, e.g., a knottin, avimer, DARPin or affinity clamp, etc. In some examples, the antibody may be a monoclonal antibody, a multispecific antibody, a bispecific antibody, a Fab2, a Fc-Fab, a camelid antibody, a peptibody scFv-Fc, an immunoglobulin, an IgG, an IgA, an IgM, an IgD, an IgE, or a combination thereof. The antibody or fragment thereof may comprise specificity to one or more components on a cell or cellular compailment, e.g., surface proteins, transmembrane proteins, post-translational modifications on proteins, polypeptides, lipids, carbohydrates, or a combination thereof. In certain cases, the antibody or fragment thereof may be engineered to comprise an affinity for a specific component on a cell or a cellular compartment. In some cases, the protein may bind to a bio marker on a cell, cellular compartment, or secreted protein. The protein may selectively bind to a biomarker on a particular cell, cellular compartment, or secreted protein. In some embodiments, the binding moiety comprises one or more antibodies or fragments thereof, combinations of antibodies, combinations of antibody fragments, or combinations of antibodies and fragments thereof.

[0060] In some embodiments, the protein binding moiety comprises a fluorescent tag. The binding moiety may comprise a fluorescent tag and another protein, e.g., an antibody or fragment thereof. The fluorescent tag may be a fluorescently-labeled antibody, a fluorescent protein, a fluorescent small molecule, a fluorescent compound, a fluorescent polypeptide, or a polynucleotide conjugated to a fluorescent polypeptide or protein. In some embodiments, the protein moiety comprises an affinity tag. The protein moiety may comprise a magnet (e.g., a magnetic particle).

[0061] The binding moiety may comprise a polypeptide. In some cases, the polypeptide may bind to one or more component on a cell, cellular compartment, or secreted protein. The polypeptide may bind to a component on a cell, cellular compailment, or secreted protein that is specific to a particular cell type. The polypeptide may bind to a component on a cell, cellular compartment, or secreted protein that is expressed on all or nearly all cell types within a given tissue. In some embodiments, the polypeptide hybridizes to the surface of a cell, cellular compartment, or secreted protein. In some embodiments, the polypeptide binding moiety comprises a fluorescent tag. In some cases, the polypeptide may bind to a biomarker on a cell, cellular compartment, or secreted protein. The polypeptide may selectively bind to a biomarker on a particular cell, cellular compailment, or secreted protein.

[0062] A pool of polypeptide binding moieties can comprise multiple groups of polypeptide binding moieties, each of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of polypeptide binding moieties in the pool. In some cases, a pool of polypeptide binding moieties comprises multiple groups of polypeptide binding moieties, some of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of polypeptide binding moieties in the pool. In some examples, a pool of polypeptide binding moieties can comprise multiple groups of polypeptide binding moieties, each of which bind to the same or similar components on a cell, cellular compartment, or secreted protein as the other groups of polypeptide binding moieties in the pool.

[0063] The binding moiety may comprise a nucleic acid molecule. In some cases, the nucleic acid molecule comprises RNA, DNA, xenonucleic acids (XNA), or a combination thereof. The nucleic acid molecule may comprise modified nucleotides, e.g., methylated or phosphorylated nucleotides. The nucleic acid molecule binding moiety may be configured to hybridize to a specific nucleic acid sequence. In some embodiments, the nucleic acid molecule may be coupled to a macromolecule, e.g., a lipid, polypeptide, protein, or carbohydrate. In some cases, the nucleic acid molecule binding moiety may bind to a cell, cellular compartment, or secreted protein. The nucleic acid molecule binding moiety may selectively bind to a biomarker on a particular cell, cellular compartment, or secreted protein.

[0064] A pool of nucleic acid molecule binding moieties can comprise multiple groups of nucleic acid molecule binding moieties, each of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of nucleic acid molecule binding moieties in the pool. In some cases, a pool of nucleic acid molecule binding moieties comprises multiple groups of nucleic acid molecule binding moieties, some of which bind to a different component on a cell, cellular compailment, or secreted protein than the other groups of nucleic acid molecule binding moieties in the pool. A pool of nucleic acid molecule binding moieties, in some cases, that comprises multiple groups of nucleic acid molecule binding moieties, each of which bind to the same or similar components on a cell, cellular compailment, or secreted protein as the other groups of nucleic acid molecule binding moieties in the pool.

[0065] In some examples, the binding moiety comprises an aptamer. The aptamer may comprise DNA, RNA, XNA, or polypeptides. In some cases, the aptamer comprises an oligonucleotide. The aptamer may be configured to target a specific molecule on a target cell. The aptamer may be configured to bind to a specific target with a specific affinity. In some cases, the aptamer may bind to one or more cell surface proteins. The aptamer may bind to a component on a cell, cellular compartment, or secreted protein that is specific to a particular cell type. The aptamer may bind to a component on a cell, cellular compartment, or secreted protein that is expressed on all or nearly all cell types within a given tissue. The aptamer may bind to a cell surface protein that is specific to a particular cell type. Alternatively, the aptamer may bind to a cell surface protein that is expressed on all or nearly all cell types within a given tissue. In some cases, the aptamer may bind to a biomarker on a cell, cellular compartment, or secreted protein. The aptamer may selectively bind to a biomarker on a particular cell, cellular compartment, or secreted protein.

[0066] A pool of aptamer binding moieties can comprise multiple groups of aptamer binding moieties, each of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups aptamer binding moieties in the pool. In some cases, a pool of aptamer binding moieties comprises multiple groups of aptamer binding moieties, some of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of aptamer binding moieties in the pool. A pool of aptamer binding moieties, in some examples, comprises multiple groups of aptamer binding moieties, each of which bind to the same or similar components on a cell, cellular compartment, or secreted protein as the other groups of aptamer binding moieties in the pool.

[0067] The binding moiety may comprise a small molecule. The small molecule may comprise a chemical or synthetic chemical. The small molecule may bind to a component on a cell, cellular compartment, or secreted protein that is specific to a particular cell type. The small molecule may bind to a component on a cell, cellular compartment, or secreted protein that is expressed on all or nearly all cell types within a given tissue. The small molecule may bind specifically to a cell surface protein that is expressed on all or nearly all cell types within a given tissue. In some cases, the small molecule may bind to a biomarker on a cell, cellular compartment, or secreted protein. The small molecule may selectively bind to a biomarker on a particular cell, cellular compartment, or secreted protein. In some embodiments, the small molecule may be coupled to a fluorescent tag.

[0068] A pool of small molecule binding moieties can comprise multiple groups of small molecule binding moieties, each of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of small molecule binding moieties in the pool. In some cases, a pool of small molecule binding moieties comprises multiple groups of small molecule binding moieties, some of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of small molecule binding moieties in the pool. A pool of small molecule binding moieties, in some examples, comprises multiple groups of small molecule binding moieties, each of which bind to the same or similar components on a cell, cellular’ compartment, or secreted protein as the other groups of small molecule binding moieties in the pool.

[0069] A pool of binding moieties can comprise one or more types of binding moiety (e.g., lipid, protein, polypeptide, aptamer, or small molecule). In some cases, a pool of binding moieties comprises multiple groups of binding moieties, each of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of binding moieties in the pool. A pool of binding moieties, in some examples, comprises multiple groups of binding moieties, some of which bind to a different component on a cell, cellular compartment, or secreted protein than the other groups of binding moieties in the pool. A pool of binding moieties can comprise multiple groups of binding moieties, each of which bind to the same or similar components on a cell, cellular compartment, or secreted protein as the other groups of binding moieties in the pool.

[0070] The method may comprise coupling the binding moiety to a cell, cellular compartment, or secreted protein. The coupling may comprise hydrogen bonding, electrostatic interactions, Van der Waal forces, hydrophobic bonds, hydrophobic interactions, covalent bonding, it- it interactions, 7t- cation interactions, or a combination thereof. In some cases, coupling of the binding moiety to a cell, cellular compartment, or secreted protein is reversible. In some examples, coupling of the binding moiety to a cell, cellular compartment, or secreted protein is irreversible. The coupling of the binding moiety to a cell, cellular’ compartment, or secreted protein may comprise affinity ligands. The affinity ligand may comprise a molecule that binds to the cell, cellular compartment, or secreted protein with an affinity that is higher than the affinity of the affinity ligand to another molecule in the tissue.

[0071] In some cases, the method comprises coupling the binding moiety to a surface moiety on the cell, cellular compartment, or secreted protein. The surface moiety may comprise a lipid, a protein, a protein comprising one or more post-translational modifications, a carbohydrate, or a combination thereof. The binding moiety may be configured to bind to a specific surface moiety on the cell, cellular compartment, or secreted protein. Moreover, the binding moiety may be configured to bind to a specific surface moiety on the cell, cellular compartment, or secreted protein with a specific affinity. The surface moiety may comprise a molecule that is transmembrane, anchored, tethered, or otherwise attached to the membrane.

[0072] In some cases, the method comprises coupling the binding moiety to a surface moiety on the cell or cellular compartment. In some examples, the surface moiety is expressed on the surface of a cell, a cellular compartment, a plasma membrane, or in a lipid bilayer. Moreover, the surface moiety can be on the surface of a cell, in or on the cell membrane, the nuclear envelope, the inner membrane of the nuclear membrane, the outer membrane of the nuclear membrane, the mitochondria, the endoplasmic reticulum, the Golgi apparatus, the plastids, the lysosomes, the vacuoles, other membrane-bound cellular compartments, or a combination thereof. In some cases, the method comprises coupling the binding moiety to a secreted protein. In some examples, the secreted protein may be a cytokine, chemokine, coagulation factor, growth factor, signaling factor, or combination thereof. In some examples the cytokine may comprise IL-10, IL-18, IL-8, IL-18, IL-3, IL-6, IL-4, IL-13, IL-11, interferon gamma, IL-15, IL-22, or IL-21. In some examples, the chemokine may comprise CCL14, CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13, CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL10. In some examples, the coagulation factor may comprise clotting factor I, clotting factor II, clotting factor III, clotting factor IV, clotting factor V, clotting factor VI, clotting factor VII, clotting factor VIII, clotting factor IX, clotting factor X, clotting factor XI, clotting factor XII, clotting factor XIII, clotting factor XIV, clotting factor XV, or clotting factor

XX.

[0073] The surface moiety may be engineered. In some cases, a cell is engineered to express a surface moiety. In some cases, a cell is engineered to heterologously express a surface moiety. In some cases, a cell is engineered to ectopically express a surface moiety. In certain embodiments, an animal is engineered to express a surface moiety in all cells or a subset of cells. In some cases, an animal is engineered to heterologously express a surface moiety. In some cases, an animal is engineered to ectopically express a surface moiety.

[0074] The binding moiety may be engineered to target an engineered variant of a surface moiety. The binding moiety may be engineered to target a population variant of a surface moiety. The binding moiety may be engineered to target a rare variant of a surface moiety. The binding moiety may be engineered to target a wild type version of a surface moiety.

[0075] In some cases, the method comprises coupling the binding moiety to a surface protein on the cell, cellular compartment, or secreted protein. In some examples, the surface protein is expressed on the surface of a cell, a cellular compartment, a plasma membrane, or in a lipid bilayer. Moreover, the surface protein can be on the surface of a cell, in or on the cell membrane, the nuclear envelope, the inner membrane of the nuclear membrane, the outer membrane of the nuclear membrane, the mitochondria, the endoplasmic reticulum, the Golgi apparatus, the plastids, the lysosomes, the vacuoles, other membrane-bound cellular compartments, or a combination thereof.

[0076] The one or more cell surface proteins may be expressed on nucleated cells. In other cases, the one or more cell surface proteins may be expressed on non-nucleated cells. In some cases, the one or more cell surface proteins is expressed on mononucleated cells. In certain embodiments, the one or more cell surface proteins is expressed on multinucleated cells. The cell surface protein may comprise a Major Histocompatibility Complex (MHC) class I molecule, a protein tyrosine phosphatase, a protein tyrosine kinase, a G-protein coupled receptor, a cluster of differentiation (CD) antigen, interleukin (IL) molecules, variants thereof, or other cell surface proteins.

[0077] In some cases, the one or more cell surface proteins is expressed on multiple cell types. The cell surface protein may be expressed on immune cells, stem cells, hematopoietic cells, endothelial cells, epithelial cells, muscle cells, nerve cells, connective tissue cells, or germ cells. The cell surface protein may comprise CD45, CD3, CD44, CD34, CDl lb, CD14, CD4, CD8, CD24, CD19, CD11, CD29, CD298 or other molecules expressed on the surface of cells.

[0078] In some cases, the one or more cell surface proteins is expressed on a specific cell type. The specific cell type may comprise an immune cell, a stem cell, a neuronal cell, a cardiac cell, a fibroblast, a blood cell, a muscle cell, a bone cell, a skin cell, an endothelial cell, an epithelial cell, an adipocyte, a germ cell, a hematopoietic cell, an erythrocyte, a leukocyte, a granulocyte, and agranulocyte, a neutrophil, and eosinophil, a basophil, a lymphocyte, a macrophage, an astrocyte, a Glial cell, oligodendrocyte, a microglial cell, an ependymal cell, a Schwann cell, a Satellite cell, a myocyte, a cardiomyocyte, a chondrocyte, an osteoblast, an osteoclast, an osteocyte, a keratinocyte, a melanocyte, a Langerhans cell, a Merkel cell, a Goblet cell, a Paneth cell, a secretory cell, a white adipocyte, a brown adipocyte, a spermatozoa, an ova, a hepatocyte, a hepatic stellate cell, a Kupffer cell, a liver sinusoidal endothelial cell, an erythroid cell, or another specific cell type. In some cases, the one or more cell surface proteins is expressed on a pathogenic or diseased cell. The one or more cell surface proteins may be expressed on a cancer cell. In certain embodiments, the one or more cell surface proteins is an indication of a pathology or disease.

[0079] The surface protein may be engineered. In some cases, a cell is engineered to express a surface protein. In some cases, a cell is engineered to heterologously express a surface protein. In some cases, a cell is engineered to ectopically express a surface protein. In certain embodiments, an animal is engineered to express a surface protein in all cells or a subset of cells. In some cases, an organism is engineered to heterologously express a surface protein. In some cases, an organism is engineered to ectopically express a surface protein.

[0080] The method disclosed herein may comprise a nucleic acid capture sequence coupled to the binding moiety. In some cases, the nucleic acid capture sequence is a universal capture sequence. A universal capture sequence as used herein refers to a nucleic acid sequence that can hybridize to a plurality of different nucleic acid molecules. A universal capture sequence can be common to nucleic acid molecules that can then hybridize to other nucleic acid molecules. The other nucleic acid molecules can comprise a common sequence to which the universal capture sequence can hybridize. In some cases, different nucleic acid molecules can comprise a common hybridization sequence to which nucleic acid molecules having the universal capture sequence can hybridize. Such nucleic acid molecules having a universal capture sequence can include nucleic acid molecules having the nucleic acid barcode sequence. In some cases, the method comprises use of multiple, nonuniversal capture sequences, e.g., the multiple capture sequences encode variability compared to one another. In certain embodiments, a binding moiety comprises a specific capture sequence that is specific to the binding moiety and distinct from other binding moieties.

[0081] The nucleic acid capture sequence may be coupled to a linker. In some embodiments, the linker is a nucleic acid sequence, a polypeptide sequence, a small molecule, a protein, or a combination thereof. The nucleic acid molecule comprising the capture sequence may be coupled to the binding moiety by a linker. In some cases, the linker comprises a linker nucleic acid sequence or a linker amino acid sequence. In some embodiments, the nucleic acid capture sequence and the nucleic acid linker are on the same nucleic acid molecule. The nucleic acid molecule comprising the capture sequence may be linked to the binding moiety by conjugation. The nucleic acid molecule comprising the capture sequence may be linked to the binding moiety by an affinity binding pair. The affinity binding pair may, for example, comprise streptavidin, avidin, and/or biotin. The nucleic acid molecule comprising the capture sequence may be covalently linked to the binding moiety. In some cases, the nucleic acid molecule comprising the capture sequence may be complexed with the binding moiety. In certain embodiments, the nucleic acid molecule comprising the capture sequence may be reversibly linked to the binding moiety.

[0082] The nucleic acid capture sequence may be coupled to one or more universal processing sequences, which are described above. In the following paragraphs, the universal processing sequence (as described above) may be referred to as a “barcode capture sequence”, which wording is intended to capture the idea that the sequence assists in capturing the sequence of a barcode in subsequent steps. The nucleic acid capture sequence may be coupled to a binding moiety nucleic acid barcode sequence that corresponds to the binding moiety. In some cases, the nucleic acid capture sequence and the binding moiety nucleic acid barcode sequence that corresponds to the binding moiety are on the same nucleic acid barcode molecule. In certain embodiments, the nucleic acid capture sequence coupled to a binding moiety nucleic acid barcode sequence that corresponds to the binding moiety is also coupled to a nucleic acid primer binding sequence. The nucleic acid capture sequence, the binding moiety nucleic acid barcode sequence, and the nucleic acid primer binding sequence may be on the same nucleic acid barcode molecule. In some cases, the nucleic acid capture sequence, the binding moiety nucleic acid barcode sequence, and the nucleic acid primer binding sequence are not on the same nucleic acid barcode molecule.

[0083] The nucleic acid barcode molecule may further comprise a barcode capture sequence. The barcode capture sequence may comprise a poly-adenosine (poly(A)) sequence. The barcode capture sequence may be a sequence that enables enrichment of the nucleic acid barcode molecule. The barcode capture sequence may comprise a reverse complement to the capture sequence coupled to the binding moiety.

[0084] In some cases, a region of the nucleic acid barcode molecule is configured to hybridize with the nucleic acid capture sequence. In some cases, the nucleic acid barcode molecule hybridizes with the nucleic acid capture sequence by a nucleic acid sequence that comprises a reverse complement to the capture sequence. In some embodiments, the nucleic acid barcode molecule is provided as a hybridized complex with the nucleic acid capture sequence. The hybridized complex may comprise the binding moiety coupled to the nucleic acid capture sequence. In some embodiments, the hybridized complex comprising the nucleic acid barcode molecule, the nucleic acid capture sequence, and the binding moiety are provided on the array. The hybridized complex comprising the nucleic acid barcode molecule, the nucleic acid capture sequence, and the binding moiety may be provided on or immobilized on the array. In some embodiments, the binding moiety is coupled to a nucleic acid capture sequence on the array. The nucleic acid molecule comprising the spatial barcode may be hybridized to the capture sequences coupled to the binding moiety already provided on the array.

[0085] In some embodiments, the array may comprise a plurality of binding moieties coupled to a nucleic acid molecule comprising a binding moiety barcode and a capture sequence. In some embodiments, the array may comprise a plurality of binding moieties coupled to a nucleic acid molecule comprising a linker and a capture sequence. The capture sequence coupled to the binding moiety may be hybridized to a reverse complement of the capture sequence in a nucleic acid molecule that further comprises a spatial barcode. The hybridization of the capture sequence to the reverse complement of the capture sequence may occur before, after, or during the placing of the tissue on the array.

[0086] In some embodiments, the array may be provided with the capture sequence coupled to the binding moiety hybridized to a nucleic acid molecule comprising a spatial barcode. In some embodiments, the array may be provided with the capture sequence coupled to the binding moiety configured to hybridize to a nucleic acid molecule comprising a spatial barcode before, after, or during the placing of the tissue on the array.

[0087] In some embodiments, the array may be provided with a nucleic acid molecule comprising a spatial barcode. In some embodiments, the array may be provided with a nucleic acid molecule comprising a spatial barcode hybridized to a capture sequence coupled to the binding moiety. In some embodiments, the array may be provided with the nucleic acid molecule comprising a spatial barcode configured to hybridize to a capture sequence coupled to the binding moiety before, after, or during the placing of the tissue on the array.

[0088] The nucleic acid barcode molecule may further comprise an adaptor sequence. Nonlimiting examples of adaptor sequences include sequencing primer binding sites, flow cell attachment sequences, index sequences, molecular index or barcode sequences, or a combination thereof.

[0089] The capture sequence and spatial barcode sequence can be assembled by polymerase chain reaction (PCR). In some embodiments, PCR can assemble adaptor or index sequences onto the nucleic acid molecule comprising the capture sequence and/or barcode sequence. In some embodiments, the capture sequence and spatial barcode sequence can be assembled by in vitro transcription. In some embodiments, the capture sequence and spatial barcode sequence can be assembled by reverse transcription.

[0090] In some cases, the assembly of the binding moiety capture sequence to the spatial barcode can be aided by template switching oligos (TSO). The assembly of the binding moiety barcode sequence to the spatial barcode can be aided by TSOs. In some embodiments, the assembly of the binding moiety capture sequence to the spatial barcode can be aided by indexing primers. The assembly of the binding moiety barcode sequence to the spatial barcode can be aided by indexing primers.

[0091] The method may comprise an array comprising a plurality of features. In some cases, the array is patterned. Alternatively, the array may be random. The array may be a microwell array. In some cases, the feature is a microwell. The microwell array may comprise a plurality of microwells. In some cases, the microwells comprise a volume of at most about IpL, at most about 2 pL, at most about 5 pL, at most about 10 pL, at most about 15 pL, at most about 20 pL, at most about 50 pL, at most about 75 pL, at most about 100 pL, at most about 150 pL, at most about 200 pL, at most about 300 pL, at most about 400 pL, at most about 500 pL, at most about 600 pL, at most about 700 pL, at most about 800 pL, at most about 900 pL, at most about InL, at most about 2 nL, at most about 5 nL, at most about 10 nL, at most about 15 nL, at most about 20 nL, at most about 50 nL, at most about 75 nL, at most about 100 nL, at most about 150 nL, at most about 200 nL, at most about 300 nL, at most about 400 nL, at most about 500 nL, at most about 600 nL, at most about 700 nL, at most about 800 nL, at most about 900 nL, at most about IpL, at most about 2pL, at most about 5pL, at most about lOpL, at most about 15pL, at most about 20pL, at most about 50pL, at most about 75qL, at most about lOOqL, at most about 150pL, at most about 200pL, at most about 300pL, at most about 400pL, at most about 500pL, at most about 600pL, at most about 700pL, at most about 800pL, at most about 900pL, at most about lOOOpL, or at most about 1500qL.

[0092] The array may comprise a plurality of features at a specific density. In some cases, the density of the plurality of features comprises at least about 96 features per array, at least about 100 features per array, at least about 384 features per array, at least about 500 features per array, at least about 1,000 features per array, at least about 5,000 features per array, at least about 10,000 features per array, at least about 100,000 features per array, at least about 1,000,000 features per array or more.

[0093] In some embodiments, each feature comprises a unique barcode. The unique barcodes may encode the location of the cells or cellular compartments. In certain embodiments, cells may be tagged with multiple unique barcodes. The multiple unique barcodes may be from one well and an adjacent well. Cells, cellular compartments, or secreted proteins comprising multiple unique barcodes may provide the location of the cell, cellular compartment, or secreted protein as being in or near multiple wells. In certain embodiments, the features may comprise more than one barcode such that the combination of the barcodes provides a unique barcode.

[0094] In some cases, diffusion of the spatial labels may provide additional resolution in identifying the location of cells within a tissue. Diffusion of the spatial label may result in a cell, cellular compartment, or secreted protein comprising multiple barcodes. In some embodiments, the multiple barcodes may be present on a cell, cellular compartment, or secreted protein at varying concentrations from one another. For example, a cell may be located at a well in an array that corresponds to a coordinate or location in the tissue, e.g., well 197 of FIG. 1. In some embodiments, the cell may be located on the right edge of well 197, in which case the cell may also comprise a barcode corresponding to well 222. In the exemplary cell, the barcode corresponding to well 222 may be present at a concentration that is equal to or less than the barcode corresponding to well 197. The presence of the two or more barcodes at different concentrations may provide information as to the location of the cell in a well. In some embodiments, the concentration of one or more nucleic acid barcode molecules is used to determine the location of the cell, cellular compartment, or secreted protein.

[0095] In yet another example, the features of the array may be permeable. In some embodiments, a micro well array may comprise permeable wells. The features of the array may comprise a matrix or features. In some embodiments, the diffusion between features may be controlled. The diffusion between features may be controlled by the density of a matrix or by the material comprising the features. In certain cases, the material comprising the microwell is permeable. In some embodiments, the material that separates an individual microwell from a second individual microwell is a hydrogel that allows for diffusion of the binding moiety and nucleic acid barcode molecule between permeable wells.

[0096] In some cases, the array is a planar surface. In some cases, the planar surface comprises a slide. In some cases, the array is a planar surface that comprises a plurality of concave features. In some cases, the array is a planar surface that comprises a plurality of convex features. In some cases, the array comprises a matrix. In some cases, the array comprises a chip. In some cases, the array comprises any surface on which a tissue or portion thereof may be mounted. The array may comprise glass, metal, plastic, or a combination thereof.

[0097] The method disclosed herein may comprise placing a tissue on an array surface (FIG. 2A). In some embodiments, the tissue is placed on or immobilized on array. A second array or a cover may also be provided (FIG. 2B). The tissue may be placed on an array or cover and contacted by the second array or cover. The tissue can be placed or sandwiched in between the two arrays or covers (FIG. 2C).

[0098] The method disclosed herein may comprise sandwiching said tissue sample between the array and another surface (FIG. 2C). The other surface may comprise an array, a slide, a cover, or a surface. In some embodiments, the other surface may comprise a similar array such as an array identical to the first array. The second array can comprise the same reagents as the first array, or may comprise different reagents such as different binding agents, different spatial barcodes, and the like. The labeling of the cells, cellular compartments, or secreted proteins may occur on both arrays, such that tissue is processed on multiple sides. In some cases, the other surface may comprise another planar surface. The other surface may comprise a cover or slide. The other surface may comprise a transparent material. The planar surface may comprise a plurality of concave features. The planar surface may comprise a plurality of convex features. In some cases, the other surface comprises a matrix. The other surface may comprise a chip. In some cases, the other surface comprises any material that can be used to sandwich a tissue or portion thereof between the other surface and an array. The other surface may comprise glass, metal, plastic, or a combination thereof. The tissue may be sandwiched between the other surface during or after the tissue is contacted to the first array.

[0099] The method may comprise the nucleic acid barcode molecule immobilized at the feature on or in the array. The nucleic acid barcode molecule may be reversibly immobilized to the feature. In certain cases, the coupling of the nucleic acid barcode molecule to the cell, cellular compartment, or secreted protein at said feature further comprises releasing the nucleic acid barcode molecule from the feature. In some embodiments, the nucleic acid barcode molecule is reversibly immobilized to the feature by a photocleavable linkage. The photocleavable linkage may be disrupted by light after the tissue is set onto the array. In a nonlimiting example (FIG. 3), a tissue 303 is placed on an array comprising a feature 300. The tissue may be covered by a second array, cover, or slip 302. A binding moiety 304 may be conjugated to a capture sequence 305. The capture sequence 305 may be hybridized to a nucleic acid barcode molecule 307. The nucleic acid barcode molecule 307 may be linked with a pho tocleav able linker 301 to a feature 300 of the array. Light 306 can be used to disrupt the photocleavable linker 301 from the feature 300. Disruption of the photocleavable linker may enable the binding moiety and the nucleic acid barcode molecule to contact the cells, cellular compartments, or secreted proteins in the tissue. The nucleic acid barcode molecule may be reversibly immobilized to the feature by a matrix. In some embodiments, the matrix comprises a hydrogel. The matrix may be permeable or impermeable.

[00100] In some examples, the feature may comprise preserved material. Preserved material may, in some cases, comprise frozen material. Preserved material may, in some cases, comprise formalin fixed paraffin embedded material. In some embodiments, the nucleic acid barcode molecule is in dry form at the feature when the tissue sample is contacted to the array. The nucleic acid molecule barcode may be suspended in fluid from the tissue sample, thereby mobilizing the nucleic acid molecular barcode. In some embodiments, the nucleic acid barcode molecule may be printed onto or into the array. The nucleic acid barcode molecule may be coupled to the binding moiety in the immobilized state.

[00101] The method as disclosed herein may further comprise enriching for a cell, cellular compartment, or secreted protein coupled to the nucleic acid barcode molecule from a plurality of cells or cellular compartments. In some embodiments, enriching for a cell, cellular compartment, or secreted protein coupled the nucleic acid barcode molecule from a plurality of cells, cellular compartments, or secreted proteins follows coupling of the nucleic acid barcode molecule to the cell, cellular compartment, or secreted protein at the feature. The nucleic acid barcode molecule may comprise a fluorescent tag. In some embodiments, enriching for a cell, cellular compartment, or secreted protein coupled the nucleic acid barcode molecule from a plurality of cells, cellular compartments, or secreted proteins may comprise selecting fluorescently labeled cells. In some embodiments, the enrichment comprises flow cytometry. In some embodiments, enriching for the cell, cellular compartment, or secreted protein coupled to the nucleic acid barcode molecule comprises affinity-based enrichment. In some embodiments, the affinity-based enrichment comprises a secondary antibody. In some embodiments, flow cytometry may be performed in combination with affinity-based enrichment. Affinity-based enrichment of the cell, cellular compartment, or secreted protein coupled to the nucleic acid barcode molecule may comprises use of streptavidin, avidin, or biotin.

[00102] In any embodiment, after the tissue sample has been contacted with the array and the nucleic acid barcode molecules have been coupled to the cells or cellular compartments, the tissue section may be separated from the array in its intact form or in pieces if it falls to pieces. In other words, the tissue section may be contacted with the array, the nucleic acid barcode molecules may couple to the cells or cellular compartments while the tissue section is still intact, and the tissue section (in its intact form) may be removed from the array (in an intact form or in pieces). As would be apparent, some of the cells in the removed tissue section will be labeled with the nucleic acid barcode molecules by this point.

[00103] As will be described in greater detail below, in any embodiment, omics data obtained from those cells can be mapped to a site in the tissue sample using the barcode sequence of the nucleic acid barcode molecule. In these embodiments, the removed tissue section is treated with a cell disassociation agent (e.g., one or more proteolytic or collagenolytic enzymes such as trypsin or Accutase, or by treating the tissue with EDTA; see, e.g., Lai Scientific Reports 2022 12: 5713) to disassociate the cells in the tissue sample. At this point, the cells may be optionally treated with a permeabilization agent, if desired, in preparation for later steps. The disassociation step should result in a solution comprising at least some single cells. Next, the method may involve subjecting the disassociated cells to a single-cell omics analysis in which analytes (genome alterations, nucleotide modifications, e.g., methyl C, histone structure, the transcriptome or protein expression) in or on the cells are detected on a cell-by-cell basis. These embodiments, involve making an ‘omics’ sequencing library (e.g., a library that allows one to read genome alterations, nucleotide modifications, e.g., methyl C, histone structure, the transcriptome and/or protein expression in the cells) from the cells after they have been disassociated. Such libraries can be made using a variety of methods, including those such as droplet-based and split-and-pool based methods commercialized by lOx Genomics, Parse Biosciences and Scale Biosciences. However the library is made, the library will contain some nucleic acids that contain an omics-related sequence (e.g., the sequence of a fragment of cDNA, genomic DNA or a barcode corresponding to a protein, for example) as well as a single-cell barcode (i.e., a barcode the identifies the cell from which the omics-related sequence is derived). After sequencing this library, omics-related sequences that are associated with same single-cell barcode can be assigned to the same cell. Thus, the single-cell barcodes allow the omics data to be assigned to cells on a cell-by-cell basis.

[00104] In some embodiments, the disassociated cells may be fixed and permeabilized after they are disassociated (e.g., using a detergent and/or a solvent) and prior to omics analysis. In other embodiments, the cells are not fixed and permeabilized after they are disassociated and prior to omics analysis.

[00105] In the present method, the nucleic acid barcode molecules added in the initial step of the method can be labeled with single-cell barcodes in the same workflow as the omics-related nucleic acids. As would be apparent, the various oligonucleotides can be designed to contain sequences that are compatible with the single cell library construction workflow and/or sequencing system being used so that the spatial barcodes can be incorporated into the library in the same workflow as the analyte-related sequences.

[00106] This results in nucleic acid molecules that contain a single-cell barcode and a barcode sequence that identifies a location of said feature on the array (the ‘spatial’ barcode). By identifying which single-cell barcode and spatial barcodes are paired, one can map the positions of the cells or cell compartments on the array. This data, in turn, provides a way to map the omics data for a cell to a location on the array (or tissue). In other words, since the location of a cell can be mapped using a spatial barcode, the omics data associated with that can be readily mapped to that location.

[00107] As explained above, the method disclosed herein may further comprise identifying the barcode sequence of the nucleic acid barcode molecule in a single cell assay. In some embodiments, the single cell assay may comprise a droplet-based assay, sequencing, cytometry, flow cytometry, mass cytometry, sorting, immunofluorescence, microscopy, morphology analysis, optics, microfluidics, or a combination thereof. A droplet-based assay may comprise single cell sequencing, single cell RNA sequencing, proteomics, chromatin accessibility, epigenetics sequencing, or a combination thereof. The method disclosed herein is agnostic to downstream assays. In some embodiments, the method is compatible with lOx Genomics, BD Rhapsody, Illumina, PacBio, Oxford Nanopore, Cytena F.Sight. Deepcell, Parse, Scale Biosciences, PipSeq and other single cell platforms.

[00108] As would be apparent, some of the sequences used in the present method, particularly a universal processing sequence, may be compatible with use in any next generation sequencing platform in which primer extension is used, e.g., Illumina’s reversible terminator method, etc. Examples of such methods are described in the following references: Margulies et al. (Nature 2005 437: 376-80); Ronaghi et al. (Analytical Biochemistry 1996 242: 84-9); Shendure et al. (Science 2005 309: 1728); Imelfort et al. (Brief Bioinform. 2009 10:609-18); Fox et al. (Methods Mol Biol. 2009;553:79-108); Appleby et al. (Methods Mol Biol. 2009;513:19-39) English et al. (PLoS One. 20127: e47768) and Morozova and Marra (Genomics. 2008 92:255-64), which are incorporated by reference for the general descriptions of the methods and the particular steps of the methods, including all starting products, reagents, and final products for each of the steps.

[00109] The sequencing step may be done using any convenient next generation sequencing method and may result in at least 10,000, at least 50,000, at least 100,000, at least 500,000, at least IM, at least 10M, at least 100M or at least IB sequence reads. In some cases, the reads arc paired-end reads.

[00110] In some cases, the method disclosed herein may further comprise, using said location of a cell, cellular compartment, or secreted protein to determine a position of an analyte of the cell, cellular compailment, or secreted protein in a tissue. Determining a position of an analyte of the cell, cellular compartment, or secreted protein in a tissue may be done after using the location of a feature of an array to identify the position of the cell, cellular compailment, or secreted protein in the tissue sample. In some embodiments, the analyte comprises a nucleic acid molecule, a protein, epigenetic information, a biomarker, a lipid, a carbohydrate, a chemical constituent, or any other molecule of interest in a subject. [00111] The method disclosed herein may comprise an enrichment. In some embodiments, the enrichment comprises enriching for the cell, cellular compartment, or secreted protein coupled to the nucleic acid barcode molecule from a plurality of cells or cellular compartments. The enrichment may occur following coupling the nucleic acid barcode molecule to the cell, cellular compartment, or secreted protein at the feature. In some cases, the enrichment enriches for live cells. The enrichment may also remove cells, cellular compartments, or secreted proteins not coupled to a nucleic acid barcode sequence, dead cells, cellular debris, other unwanted biomaterials, buffers, salts, metals, volume, or a combination thereof. In certain embodiments, the enrichment selects for cells that are labeled with a fluorescent marker. In some cases, the fluorescent marker comprises a fluorescent antibody. The fluorescent antibody may be specific to a particular cell surface marker. In some cases, the fluorescent antibody may be specific to a binding moiety. In some cases, the enrichment comprises affinity-based enrichment. The affinity-based enrichment may comprise use of streptavidin, avidin, and/or biotin.

[00112] The method disclosed herein can be applied to a range of sample types. In some embodiments, the sample comprises a whole organism. In some embodiments, the sample comprises a whole organ. In some embodiments, the sample comprises a tissue sample. In some embodiments, the tissue sample comprises a whole tissue, a tissue section, a cell monolayer, an organoid, a fixed cell, or any combination thereof. In some embodiments, the sample may be embedded in a matrix. In some embodiments, the sample may be embedded in a hydrogel. In some embodiments, the hydrogel may comprise polyacrylamide, collagen, fibrin, alginate, polyethylene glycol, hyaluronic acid, polypeptides, or a combination thereof. The cells, cellular compartments, or secreted proteins within the tissue sample may be dissociated from the tissue. In some embodiments, dissociating the tissue into single cells comprising a cell, cellular compartment, or secreted protein occurs during or after the tissue is contacted to the array. In certain cases, dissociating the tissue into single cells comprising a cell, cellular compartment, or secreted protein occurs during or after the nucleic acid barcode molecule is coupled to the cell, cellular compartment, or secreted protein. The dissociating may be done at the feature of the array. Kits

[00113] Also provided by this disclosure are kits for practicing the subject methods, as described above. Descriptions of the reagents that can be found in a kit are described above. The various components of the kit may be present in separate containers or certain compatible components may be pre-combined into a single container, as desired.

[00114] In some embodiments, a kit may comprise an array of features, wherein a plurality of the features (e.g., at least 96, at least 384, at least 1,000, at least 10,000, at least 50,000 or at least 100,000 features) each comprise one or more nucleic acid barcode molecules that comprise: (a) a non-nucleotidyl moiety that binds to cells or a subcellular compartment thereof (e.g., a lipid such as a fatty acid (e.g., lignoceric acid or palmitic acid) and/or cholesterol that inserts into a membrane, or a proteinaceous binding agent (such as an antibody) that recognizes a protein or other biomolecule in or on the cells , and (b) a spatial barcode sequence that alone or in combination with one or more other spatial barcode sequences in a feature identifies a location of the feature on the array.

[00115] In some embodiments, the nucleic acid barcode molecule may comprise: (i) a first oligonucleotide that comprises a capture sequence of at least 12 (e.g., at least 15 or at least 20) nucleotides and the non-nucleotidyl moiety; and (ii) a second oligonucleotide comprising a sequence that is complementary to the capture sequence of the first oligonucleotide and the spatial barcode sequence. In these embodiments, the first and second oligonucleotides may be pre-hybridized together. However, this may not be necessary because the oligonucleotides may hybridize together in the coupling step, i.e., during use. In these embodiments, in the first oligonucleotide, the capture sequence and the non-nucleotidyl moiety may be linked to each other noncovalently (e.g., via a biotin/avidin linkage) or covalently. In some of these embodiments, the first oligonucleotide may be the same in all features of the plurality of features and/or the spatial barcode sequences may vary from feature to feature. In any embodiment, the second oligonucleotide may further comprise one or more universal processing sequences, wherein the one or more universal processing sequences may be the same in all features of the array.

[00116] In any embodiment, the array may comprise at least 100 of the features. The features may be wells or, in some embodiments, the array may planar and the nucleic acid barcode molecules are on a planar surface. The nucleic acid barcode molecules may be in dry form in or on the array or in solution.

[00117] As noted above, in some embodiments, the non-nucleotidyl moiety is a lipid that inserts into biological membranes or a protein such as an antibody. In some embodiments, the non-nucleotidyl moiety may bind to the surface of cells, whereas in other embodiments, it may bind to a subcellular compartment, e.g., nuclei. The nucleic acid barcode molecule may be in dried or liquid form in the array.

[00118] In addition to the above-mentioned components, the subject kit may further include instructions for using the components of the kit to practice the subject method.

Computer systems

[00119] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 8 shows a computer system 801 that is programmed or otherwise configured to generate, interpret, and store data sets. The computer system 801 can regulate various aspects of the method of the present disclosure, such as, for example, designing an array, designing sequences, identifying and designing binding moieties, generating data, interpreting data, and storing data. The computer system 801 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[00120] The computer system 801 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 805, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 801 also includes memory or memory location 810 (e.g., random-access memory, read-only memory, Hash memory), electronic storage unit 815 (e.g., hard disk), communication interface 820 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 825, such as cache, other memory, data storage and/or electronic display adapters. The memory 810, storage unit 815, interface 820 and peripheral devices 825 are in communication with the CPU 805 through a communication bus (solid lines), such as a motherboard. The storage unit 815 can be a data storage unit (or data repository) for storing data. The computer system 801 can be operatively coupled to a computer network (“network”) 830 with the aid of the communication interface 820. The network 830 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet. The network 830 in some cases is a telecommunication and/or data network. The network 830 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 830, in some cases with the aid of the computer system 801, can implement a peer-to-peer network, which may enable devices coupled to the computer system 801 to behave as a client or a server.

[00121] The CPU 805 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 810. The instructions can be directed to the CPU 805, which can subsequently program or otherwise configure the CPU 805 to implement methods of the present disclosure. Examples of operations performed by the CPU 805 can include fetch, decode, execute, and writeback.

[00122] The CPU 805 can be pail of a circuit, such as an integrated circuit. One or more other components of the system 801 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[00123] The storage unit 815 can store files, such as drivers, libraries and saved programs. The storage unit 815 can store user data, e.g., user preferences and user programs. The computer system 801 in some cases can include one or more additional data storage units that are external to the computer system 801, such as located on a remote server that is in communication with the computer system 801 through an intranet or the Internet.

[00124] The computer system 801 can communicate with one or more remote computer systems through the network 830. For instance, the computer system 801 can communicate with a remote computer system of a user (e.g., provide updates, reports, errors, or troubleshooting). Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android- enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 801 via the network 830. [00125] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 801, such as, for example, on the memory 810 or electronic storage unit 815. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 805. In some cases, the code can be retrieved from the storage unit 815 and stored on the memory 810 for ready access by the processor 805. In some situations, the electronic storage unit 815 can be precluded, and machine-executable instructions are stored on memory 810.

[00126] The code can be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.

[00127] Aspects of the systems and methods provided herein, such as the computer system 801, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non- transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[00128] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[00129] The computer system 801 can include or be in communication with an electronic display 835 that comprises a user interface (UI) 840 for providing, for example, information, updates, quality control information, data, errors, options, troubleshooting, or other information that a user may access. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[00130] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 805. The algorithm can, for example, generate and read sequencing information, base call, align, identify errors, troubleshoot errors, generate statistics, calculate quality control information, generate files, or a combination thereof.

EMBODIMENTS

[00131] Embodiment 1. A method for identifying a position of a cell or cellular compartment of a tissue sample, comprising:

[00132] (a) providing an array comprising a plurality of features, wherein a feature of the plurality of features comprises a nucleic acid barcode molecule comprising a barcode sequence, wherein said barcode sequence identifies a location of said feature on said array;

[00133] (b) contacting said tissue sample with said array, wherein, said cell or cellular compartment of said tissue sample is positioned at said feature on said array upon said contacting;

[00134] (c) coupling said nucleic acid barcode molecule to said cell or cellular compartment at said feature;

[00135] (d) identifying said barcode sequence of said nucleic acid barcode molecule, thereby identifying said location of said feature of said array; and

[00136] (e) using said location of said feature of said array to identify said position of said cell or cellular compailment in said tissue sample.

[00137] Embodiment 2. The method of embodiment 1, wherein said feature comprising a nucleic acid barcode molecule further comprises a binding moiety.

[00138] Embodiment 3. The method of embodiment 2, wherein said binding moiety comprises a lipid.

[00139] Embodiment 4. The method of embodiment 2, wherein said binding moiety comprises a protein.

[00140] Embodiment 5. The method of embodiment 4, wherein said protein comprises an antibody or a fragment of an antibody.

[00141] Embodiment 6. The method of embodiment 2, wherein (c) comprises coupling said binding moiety to said cell or cellular compartment. [00142] Embodiment 7. The method of embodiment 6, wherein (c) comprises coupling said binding moiety to a surface protein on said cell or cellular compartment.

[00143] Embodiment 8. The method of any one of embodiments 2-7, wherein said binding moiety is coupled to a nucleic acid capture sequence, and wherein said nucleic acid barcode molecule is configured to hybridize with said nucleic acid capture sequence.

[00144] Embodiment 9. The method of embodiment 8, wherein said nucleic acid barcode molecule further comprises a poly-adenosine sequence.

[00145] Embodiment 10. The method of any one of embodiments 2-9, wherein said array is a microwell array and said feature is a microwell.

[00146] Embodiment 11. The method of any one of embodiments 2-10, wherein said array comprises a planar surface.

[00147] Embodiment 12. The method of any one of embodiments 2-11, wherein, during or after (b), said tissue sample is sandwiched between said array and another surface.

[00148] Embodiment 13. The method of embodiment 12, wherein said other surface is another array.

[00149] Embodiment 14. The method of any one of embodiments 1-13, wherein said nucleic acid barcode molecule is immobilized at said feature.

[00150] Embodiment 15. The method of any one of embodiments 1-14, wherein said nucleic acid barcode molecule is reversibly immobilized to said feature.

[00151] Embodiment 16. The method of embodiment 15, wherein (c) comprises releasing said nucleic acid barcode molecule from said feature.

[00152] Embodiment 17. The method of embodiment 16, wherein said nucleic acid barcode molecule is reversibly immobilized to said feature through a photocleavable linkage.

[00153] Embodiment 18. The method of embodiment 15, wherein said nucleic acid barcode molecule is reversibly immobilized to said feature through a matrix.

[00154] Embodiment 19. The method of embodiment 18, wherein said matrix comprises a hydrogel.

[00155] Embodiment 20. The method of embodiment 1, wherein in (a) said nucleic acid barcode molecule is in dry form at said feature. [00156] Embodiment 21. The method of embodiment 20, further comprising, following (a), suspending said nucleic acid barcode molecule in fluid from said tissue sample.

[00157] Embodiment 22. The method of embodiment 1, wherein said feature comprises preserved material.

[00158] Embodiment 23. The method of embodiment 22, wherein said preserved material comprises frozen material.

[00159] Embodiment 24. The method of any one of embodiments 1-23, further comprising, following (c), enriching for said cell or cellular compailment coupled to said nucleic acid barcode molecule from a plurality of cells or cellular compartments.

[00160] Embodiment 25. The method of embodiment 24, wherein enriching for said cell or cellular compartment coupled to said nucleic acid barcode molecule comprises flow cytometry.

[00161] Embodiment 26. The method of embodiment 24, wherein said feature comprises a fluorescent tag.

[00162] Embodiment 27. The method of embodiment 24, wherein enriching for said cell or cellular feature coupled to said nucleic acid barcode molecule comprises affinity-based enrichment.

[00163] Embodiment 28. The method of embodiment 24, wherein said affinity-based enrichment comprises use of streptavidin, avidin, or biotin.

[00164] Embodiment 29. The method of any one of embodiments 1-28, further comprising, during or after (d), identifying said barcode sequence of said nucleic acid barcode molecule in a single cell assay.

[00165] Embodiment 30. The method of embodiment 29, wherein said single cell assay comprises nucleic acid sequencing.

[00166] Embodiment 31. The method of embodiment 29, wherein said single cell assay comprises flow cytometry.

[00167] Embodiment 32. The method of embodiment 29, wherein said single cell assay comprises mass cytometry. [00168] Embodiment 33. The method of any one of embodiments 1-32, further comprising, during or after (d), determining said location of said cell or cellular compartment by determining a concentration of said nucleic acid barcode.

[00169] Embodiment 34. The method of any one of embodiments 1-33, wherein said tissue sample comprises a tissue section, a cell monolayer, an organoid, a fixed cell, or any combination thereof.

[00170] Embodiment 35. The method of any one of embodiments 1-34, further comprising, during or after (b), dissociating said tissue into single cells comprising said cell or cellular compartment.

[00171] Embodiment 36. The method of any one of embodiments 1-34, further comprising, during or after (c), dissociating said tissue into single cells comprising said cell or cellular compartment.

[00172] Embodiment 37. The method of any one of embodiments 1-36, further comprising, during or after (e), using said location of said cell or cellular compartment to determine a position of an analyte of said cell or cellular compartment in said tissue.

[00173] Embodiment 38. A method for identifying a position of a secreted protein of a tissue sample, comprising:

[00174] a. providing an array comprising a plurality of features, wherein a feature of the plurality of features comprises a nucleic acid barcode molecule comprising a barcode sequence, wherein said barcode sequence identifies a location of said feature on said array;

[00175] b. contacting said tissue sample with said array, wherein, said secreted protein of said tissue sample is positioned at said feature on said array upon said contacting;

[00176] c. coupling said nucleic acid barcode molecule to said secreted protein at said feature;

[00177] d. identifying said barcode sequence of said nucleic acid barcode molecule, thereby identifying said location of said feature of said array; and

[00178] e. using said location of said feature of said array to identify said position of said secreted protein in said tissue sample.

[00179] Embodiment 39. The method of embodiment 38, wherein said feature comprising a nucleic acid barcode molecule further comprises a binding moiety.

[00180] Embodiment 40. The method of embodiment 39, wherein said binding moiety comprises a lipid. [00181] Embodiment 41. The method of embodiment 39, wherein said binding moiety comprises a protein.

[00182] Embodiment 42. The method of embodiment 41, wherein said protein comprises an antibody or a fragment of an antibody.

[00183] Embodiment 43. The method of embodiment 39, wherein (c) comprises coupling said binding moiety to said secreted protein.

[00184] Embodiment 44. The method of any one of embodiments 38-43, wherein said binding moiety is coupled to a nucleic acid capture sequence, and wherein said nucleic acid barcode molecule is configured to hybridize with said nucleic acid capture sequence.

[00185] Embodiment 45. The method of embodiment 44, wherein said nucleic acid barcode molecule further comprises a poly-adenosine sequence.

[00186] Embodiment 46. The method of any one of embodiments 39-45, wherein said array is a microwell array and said feature is a microwell.

[00187] Embodiment 47. The method of any one of embodiments 39-46, wherein said array comprises a planar surface.

[00188] Embodiment 48. The method of any one of embodiments 39-47, wherein, during or after (b), said tissue sample is sandwiched between said array and another surface.

[00189] Embodiment 49. The method of embodiment 48, wherein said other surface is another array.

[00190] Embodiment 50. The method of any one of embodiments 38-49, wherein said nucleic acid barcode molecule is immobilized at said feature.

[00191] Embodiment 51. The method of any one of embodiments 38-50, wherein said nucleic acid barcode molecule is reversibly immobilized to said feature.

[00192] Embodiment 52. The method of embodiment 51, wherein (c) comprises releasing said nucleic acid barcode molecule from said feature.

[00193] Embodiment 53. The method of embodiment 52, wherein said nucleic acid barcode molecule is reversibly immobilized to said feature through a photocleavable linkage.

[00194] Embodiment 54. The method of embodiment 51, wherein said nucleic acid barcode molecule is reversibly immobilized to said feature through a matrix. [00195] Embodiment 55. The method of embodiment 51, wherein said matrix comprises a hydrogel.

[00196] Embodiment 56. The method of embodiment 38, wherein in (a) said nucleic acid barcode molecule is in liquid form at said feature.

[00197] Embodiment 57. The method of embodiment 38, further comprising, following (a), suspending said nucleic acid barcode molecule in fluid from said tissue sample.

[00198] Embodiment 58. The method of embodiment 38, wherein said feature comprises preserved material.

[00199] Embodiment 59. The method of embodiment 58, wherein said preserved material comprises frozen material.

[00200] Embodiment 60. The method of any one of embodiments 38-59, further comprising, following (c), enriching for said secreted protein coupled to said nucleic acid barcode molecule from a plurality of secreted proteins.

[00201] Embodiment 61. The method of embodiment 60, wherein enriching for said secreted protein coupled to said nucleic acid barcode molecule comprises flow cytometry.

[00202] Embodiment 62. The method of embodiment 60, wherein said feature comprises a fluorescent tag.

[00203] Embodiment 63. The method of embodiment 60, wherein enriching for said secreted protein coupled to said nucleic acid barcode molecule comprises affinity-based enrichment.

[00204] Embodiment 64. The method of embodiment 60, wherein said affinity-based enrichment comprises use of streptavidin, avidin, or biotin.

[00205] Embodiment 65. The method of any one of embodiments 38-64, further comprising, during or after (d), identifying said barcode sequence of said nucleic acid barcode molecule in a single cell assay.

[00206] Embodiment 66. The method of embodiment 65, wherein said single cell assay comprises nucleic acid sequencing.

[00207] Embodiment 67. The method of embodiment 65, wherein said single cell assay comprises flow cytometry. [00208] Embodiment 68. The method of embodiment 65, wherein said single cell assay comprises mass cytometry.

[00209] Embodiment 69. The method of any one of embodiments 38-68, further comprising, during or after (d), determining said location of said secreted protein by determining a concentration of said nucleic acid barcode.

[00210] Embodiment 70. The method of any one of embodiments 38-69, wherein said tissue sample comprises a tissue section, a cell monolayer, an organoid, a fixed cell, or any combination thereof.

[00211] Embodiment 71. The method of any one of embodiments 38-70, further comprising, during or after (b), dissociating said tissue into single cells comprising said secreted protein.

[00212] Embodiment 72. The method of any one of embodiments 38-70, further comprising, during or after (c), dissociating said tissue into single cells comprising said secreted protein.

[00213] Embodiment 73. The method of any one of embodiments 38-72, further comprising, during or after (e), using said location of said secreted protein to determine a position of an analyte of said secreted protein in said tissue.

[00214] Embodiment 74. A method for labeling cells or a subcellular compartment thereof, comprising:

[00215] (a) providing an array comprising a plurality of features each comprising one or more nucleic acid barcode molecules that comprise:

[00216] (!) a non-nucleotidyl moiety that binds to cells or a subcellular compartment thereof, and

[00217] (ii) a spatial barcode sequence that alone or in combination with one or more other spatial barcode sequences in a feature identifies a location of the feature on the array;

[00218] (b) contacting a tissue sample with the array and, while the tissue sample is contacted with the array, allowing the nucleic acid barcode molecules to couple to the cells or a subcellular compartment thereof in the tissue sample;

[00219] (c) removing the tissue sample from the array; and

[00220] (d) disassociating the cells or subcellular compartments thereof in the tissue sample after it has been removed from the array to produce a suspension comprising single cells or subcellular compartments that are labeled with one or more spatial barcode sequences that identifies a location of a feature on the array.

[00221] Embodiment 75. The method of embodiment 74, further comprising:

[00222] (e) making a single-cell sequencing library from the suspension, a subset of cells thereof, or a subcellular compartment of either;

[00223] (f) sequencing the single-cell sequencing library to produce sequence reads;

[00224] (g) identifying one or more spatial barcode sequences in the sequence reads, and

[00225] (h) mapping the location of a cell or subcellular compartment to a feature of the array using the one or more spatial barcode sequences.

[00226] Embodiment 76. The method of embodiment 75, wherein:

[00227] in (e) the single-cell sequencing library is an omics library that comprises:

[00228] (i) polynucleotides that comprise an analyte -related nucleic acid linked to a cell-specific barcode; and

[00229] (ii) polynucleotides that comprise a spatial barcode linked to a cell-specific barcode;

[00230] the sequencing step of (f) produces omics data;

[00231] and the method further comprises mapping the omics data for an individual cell or cellular compartment to a feature of the array using the one or more spatial barcodes for the individual cell or cellular compartment.

[00232] Embodiment 77. The method of embodiment 76, further comprising:

[00233] reconstructing an image of the tissue sample using the mapped omics data.

[00234] Embodiment 78. The method of any one of embodiments 74-77, wherein the non-nucleotidyl moiety of (a)(i) is:

[00235] a binding agent that recognizes a protein, or

[00236] a lipid that integrates into a membrane.

[00237] Embodiment 79. The method of embodiment 78, wherein the non-nucleotidyl moiety is an antibody.

[00238] Embodiment 80. The method of embodiment 78, wherein the non-nucleotidyl moiety is a fatty acid or cholesterol. [00239] Embodiment 81. The method of any prior embodiment, wherein a nucleic acid barcode molecule comprises:

[00240] (i) a first oligonucleotide that comprises a capture sequence and the non-nucleotidyl moiety;

[00241] (ii) a second oligonucleotide comprising a sequence that is complementary to the capture sequence of the first oligonucleotide, and the spatial barcode.

[00242] Embodiment 82. The method of embodiment 81 , wherein the second oligonucleotide further comprises one or more universal processing sequences.

[00243] Embodiment 83. The method of any prior embodiment, wherein the array is an array of microwells and each feature is a microwell.

[00244] Embodiment 84. The method of any prior embodiment, wherein the array is planar and the nucleic acid barcode molecules are on a planar surface.

[00245] Embodiment 85. The method of any prior embodiment, further comprising enriching for cells or a subcellular compartment from the suspension.

[00246] Embodiment 86. The method of any prior embodiment, wherein said tissue sample comprises a tissue section, a cell monolayer or an organoid and the tissue sample is fresh, frozen, fixed and/or permeabilized.

[00247] Embodiment 87. The method of any one of embodiments 75-86, wherein the single-cell sequencing library of (e) comprises cDNA, genomic DNA, probe sequences or ligation products containing the same, or barcodes that identify proteins or antibodies.

[00248] Embodiment 88. The method of any prior embodiment, wherein the non-nucleotidyl moiety binds to the surface of cells and the suspension produced in (d) comprises single cells.

[00249] Embodiment 89. The method of any prior embodiment, wherein the non-nucleotidyl moiety binds to a subcellular compartment, and wherein the tissue sample is optionally fixed and/or permeabilized prior to step (b) to allow the non-nucleotidyl moiety into the cells, and wherein the suspension produced in (d) comprises single cells or subcellular compailments.

[00250] Embodiment 90. The method of embodiment 89, wherein subcellular compartments are nuclei. [00251] Embodiment 91. The method of any prior embodiment, wherein the nucleic acid barcode molecule of (a) comprises a fluorescent moiety, and wherein the method comprises detecting the fluorescent moiety in the tissue sample, between steps (c) and (d).

[00252] Embodiment 92. The method of any prior embodiment, wherein, in (b), the tissue sample is sandwiched between two of the arrays, thereby allowing the nucleic acid barcode molecules from both arrays to couple to the cells or subcellular compartments thereof and, in step (c) the tissue sample is removed from both arrays.

[00253] Embodiment 93. The method of any prior embodiment, wherein, in (a), the nucleic acid barcode molecules are in solution.

[00254] Embodiment 94. The method of any one of embodiments 74-92, wherein the nucleic acid barcode molecules of (a) are in dried form, and become hydrated upon contact with the tissue sample in step (b).

[00255] Embodiment 95. The method of any prior embodiment, wherein the nucleic acid barcode molecules of (a) are tethered to the array and step (b) comprises releasing the nucleic acid barcode molecules from the array.

[00256] Embodiment 96. A kit comprising an array of features, wherein a plurality of the features each comprise

[00257] one or more nucleic acid barcode molecules that comprise:

[00258] (a) a non-nucleotidyl moiety that binds to cells or a subcellular compartment thereof, and

[00259] (b) a spatial barcode sequence that alone or in combination with one or more other spatial barcode sequences in a feature identifies a location of the feature on the array.

[00260] Embodiment 97. The kit of embodiment 96, wherein a nucleic acid barcode molecule comprises:

[00261] (i) a first oligonucleotide that comprises a capture sequence and the non-nucleotidyl moiety; and

[00262] (ii) a second oligonucleotide comprising a sequence that is complementary to the capture sequence of the first oligonucleotide and the spatial barcode sequence. [00263] Embodiment 98. The kit of embodiment 97, wherein the first oligonucleotide of (i) is the same in all features of the plurality of features.

[00264] Embodiment 99. The kit of embodiment 96 or 97, wherein the spatial barcode sequences vary from feature to feature in the plurality of features.

[00265] Embodiment 100. The kit of any of embodiments 97-99 wherein the second oligonucleotide further comprises one or more universal processing sequences, wherein the one or more universal processing sequences are the same in all features of the plurality of features.

[00266] Embodiment 101. The kit of any of embodiments 96-100, wherein the plurality of features comprises at least 100 of the features.

[00267] Embodiment 102. The kit of any of embodiments 96-101, wherein the features are wells.

[00268] Embodiment 103. The kit of any one of embodiments 96-101, wherein the array is planar and the nucleic acid barcode molecules are on a planar surface.

[00269] Embodiment 104. The kit of any one of embodiments 96-103, wherein the nucleic acid barcode molecules are in dry form.

[00270] Embodiment 105. The kit of any one of embodiments 96-104, wherein the nonnucleotidyl moiety is a lipid that inserts into biological membranes.

[00271] Embodiment 106. The kit of any one of embodiments 96-104, wherein the nonnucleotidyl moiety is an antibody.

[00272] Embodiment 107. The kit of any one of embodiments 96-106, wherein the non-nucleotidyl moiety binds to the surface of cells.

[00273] Embodiment 108. The kit of any one of embodiments 96-106, wherein the non-nucleotidyl moiety binds to a subcellular compartment.

[00274] Embodiment 109. The kit of any one of embodiments 108, wherein the non-nucleotidyl moiety binds to nuclei.

EXAMPLES

Example 1. Spatially labeling cells of a tissue [00275] To spatially label cells of a tissue, a microwell array is provided to include binding moieties coupled to a spatial barcode (FIGs. 4A and 4B). In this example, each well of the micro well array includes an antibody binding moiety and a spatial barcode (FIG. 4A). More specifically, each well of the microwell array includes an antibody conjugated to a first nucleic acid molecule that comprises a universal capture sequence, an antibody barcode sequence, and a first primer binding sequence (FIG. 4B). The first nucleic acid molecule is hybridized to a second nucleic acid molecule that comprises the reverse complement of the universal capture sequence, a second primer binding sequence, a spatial barcode sequence, and a poly(A) sequence (FIG. 4B). The antibodies in a single microwell include a mixture of antibodies against MHC Class I and CD45 each coupled to a barcode corresponding to the same microwell. The antibody-barcode complex is printed in liquid form into the micro well and then allowed to dry.

[00276] For example, the tissue comprises liver slices harvested from a subject, sectioned, frozen, and placed onto the microwell array over dry ice using forceps. In this example, a second array is introduced on the opposite side of the liver, and the liver will be sandwiched between the two arrays. Pressure is applied to the second array to seal the tissue in the array. The fluid from the tissue resuspends the dried antibody-barcode complexes immobilized in the microwells. Once in solution, the antibodies bind to Major Histocompatibility Complex (MHC) Class I or CD45 on the surface of the nucleated cells and leukocytes in the liver, respectively. Once sealed, the array will be placed in a swing bucket centrifuge set to 50 RCF for 1 minute at 4°C. After centrifugation, the array may optionally be incubated for 30 to 45 minutes at 37°C to 42°C with agitation to dissociate the liver tissue.

[00277] The top array will then be removed and set aside face up so any liquid/tissue adhered is uncontaminated. The microwell array chip is held nearly perpendicular over the reagent reservoir so that liquid can drip down into the reservoir. Using a 200 pL pipette, 200 pL of the cell extraction buffer is withdrawn from the reservoir and “shot” into the wells of the chip to extract out tissue and cells, resuspending them and dripping them into the reagent reservoir. The buffer is introduced nearly perpendicular relative to the microwell array surface. The rinse is repeated several times, reusing the same buffer in the reservoir. [00278] The process is repeated with the top array, introducing buffer onto the surface of the slide (or gasket) to resuspend tissue and cells that have loosely adhered and allowing them to drip into the reagent reservoir. All the cells and tissue are to be now present in the reagent reservoir. The mix is slowly pipetted lOx to mechanically break up larger tissue chunks and release cells. Using the same pipette, the cells are strained through a 70 pm cell strainer to remove any intact tissue chunks.

[00279] The cells are washed using the swing bucket centrifuge set to 4°C, and the tube containing the cells is spun down at 500g for 5 minutes. The supernatant is aspirated and the cells are resuspended in 1 mL of PBS + 0.04% BSA. The wash is repeated two times. The final suspension is completed in a volume and buffer necessary and readied for analysis in a single cell assay.

Example 2. Analyzing spatially labeled cells in a single-cell assay

[00280] The labeled cells prepared in example 1 are then processed in a single cell assay. As an example, the single cell assay is a droplet-based assay. Labeled cells from example 1 are separated into individual droplets (FIG. 5A) each comprising a labeled cell and droplet nucleic acid barcode molecules. In this example, although not required, droplet nucleic acid barcode molecules can be provided in the droplet coupled to a gel bead. The labeled cell comprises the binding moiety (for example, a mixture of MHC Class I antibodies and CD45 antibodies) conjugated to a nucleic acid molecule comprising a linker and a capture sequence. The gel bead comprises nucleic acid molecules that comprise a primer binding site, a droplet barcode sequence, a unique molecular identifier (UMI) and a capture sequence or poly(dT) sequence. In the droplets (FIGs. 5B and 5C), the nucleic acid molecules from the gel bead hybridize to a complementary sequence from the labeled cell. Before, during or after barcoding, the nucleic acid molecules of the gel bead can be released from the gel bead. For example, the poly(dT) sequences from the gel bead hybridize to the poly(A) sequences in the nucleic acid molecule comprising the spatial barcode (FIG. 5B), or to the poly(A) tails of mRNA sequences from the cell (FIG. 5C). In the droplets, reverse transcriptase (RT) transcribes nucleic acid molecules that comprise the droplet barcode and the spatial barcode. The RT also reverse-transcribes nucleic acid molecules that comprise the droplet barcode and gene transcript. [00281] The nucleic acid molecules transcribed by RT are amplified using a high-fidelity PCR with dual index primers. The amplicons are prepared for sequencing using a library preparation kit. The library is run on a short-read sequencing machine and the sequences of each molecule are determined. Some sequences include a droplet barcode and a spatial barcode; these molecules determine which spatial barcode corresponds with which droplet. To determine which transcripts correspond with a cell in a particular location, the molecules with a droplet barcode are matched to the spatial barcode that is matched on the molecules that comprise the droplet barcode and the spatial barcode. The transcriptome and spatial location are then determined for the cells in the tissue.

Example 3. Analyzing spatially labeled cells in a single-cell assay

[00282] The labeled cells prepared in example 1 are then processed in a single cell assay. As an example, the single cell assay is a microwell based assay. Labeled cells from example 1 are separated into individual microwells, each comprising a labeled cell and microwell nucleic acid barcode molecules. In this example, although not required, micro well nucleic acid barcode molecules can be provided in the micro well. In this example, although not required, the micro well may comprise a bead. The labeled cell comprises the binding moiety (for example, a mixture of MHC Class I antibodies and CD45 antibodies) conjugated to a nucleic acid molecule comprising a linker and a capture sequence. The microwell comprises nucleic acid molecules that comprise a primer binding site, a microwell barcode sequence, a unique molecular identifier (UMI) and a capture sequence or poly(dT) sequence. The microwell can comprise a bead that comprises nucleic acid molecules that comprise a primer binding site, a microwell barcode sequence, a unique molecular identifier (UMI) and a capture sequence or poly(dT) sequence. In the microwell, the nucleic acid molecules hybridize to a complementary sequence from the labeled cell. For example, the poly(dT) sequences from the microwell hybridizes to the poly(A) sequences in the nucleic acid molecule comprising the spatial barcode, or to the poly(A) tails of mRNA sequences from the cell. In the microwell, reverse transcriptase (RT) transcribes nucleic acid molecules that comprise the microwell barcode and the spatial barcode. The RT also reverse-transcribes nucleic acid molecules that comprise the microwell barcode and gene transcript. [00283] The nucleic molecules transcribed by RT are amplified using a high-fidelity PCR with dual index primers. The amplicons are prepared for sequencing using a library preparation kit. The library is run on a short-read sequencing machine and the sequences of each molecule are determined. Some sequences include a microwell barcode and a spatial barcode; these molecules determine which spatial barcode corresponds with which droplet. To determine which transcripts correspond with a cell in a particular location, the molecules with a microwell barcode are matched to the spatial barcode that is matched on the molecules that comprise the microwell barcode and the spatial barcode. The transcriptome and spatial location are then determined for the cells in the tissue.

Example 4. Spatially Analyzing Transcriptomes, Proteomes, and Set of Proteins

[00284] To determine the location of cells in a tissue, a microwell array is provided to include binding moieties coupled to a spatial barcode (FIG. 6). In this example, each well of the microwell array includes a mixture of antibody binding moieties, each of which is conjugated to a spatial barcode corresponding to the array feature or well (FIG. 6). More specifically, each well of the microwell array includes an antibody conjugated to a first nucleic acid molecule that comprises a universal capture sequence, an antibody barcode sequence, and a first primer binding sequence (FIG. 6). The first nucleic acid molecule is hybridized to a second nucleic acid molecule that comprises the reverse complement of the universal capture sequence, a second primer binding sequence, a spatial barcode sequence, and a poly (A) sequence (FIG. 6). The antibodies in a single microwell include a mixture of antibodies against target proteins, including MHC Class 1, CD45, CD3 and CD27, each coupled to a barcode corresponding to the same microwell. The antibodybarcode complex is printed in liquid form into the micro well and allowed to dry.

[00285] For example, the tissue comprises liver harvested from a subject, sectioned, frozen, and placed onto the microwell array over dry ice using forceps. In this example, a second array is introduced on the opposite side of the liver, and the liver will be sandwiched between the two arrays. Pressure is applied to the second array to seal the tissue in the array. The fluid from the tissue resuspends the dried antibody-barcode complexes immobilized in the micro wells. Once in solution, the antibodies bind the array of cell surface proteins on cells expressing the target protein. Once sealed, the array will be placed in a swing bucket centrifuge set to 50 RCF for 1 minute at 4°C. After centrifugation, the array will be incubated for 30 to 45 minutes at 37°C to 42°C with agitation to dissociate the liver tissue.

[00286] The top array will then be removed and set aside face up so any liquid/tissue adhered is uncontaminated. The microwell array chip is held nearly perpendicular over the reagent reservoir so that liquid can drip down into the reservoir. Using a 200 pL pipette, 200 p L of the cell extraction buffer is withdrawn from the reservoir and “shot” into the wells of the chip to extract out tissue and cells, resuspending them and dripping them into the reagent reservoir. The buffer is introduced nearly perpendicular relative to the microwell array surface. The rinse is repeated several times, reusing the same buffer in the reservoir.

[00287] The process is repeated with the top array, introducing buffer onto the surface of the slide (or gasket) to resuspend tissue and cells that have loosely adhered and allowing them to drip into the reagent reservoir. All the cells and tissue are to be now present in the reagent reservoir. The mix is slowly pipetted lOx to mechanically break up larger tissue chunks and release cells. Using the same pipette, the cells are strained through a 70 pm cell strainer to remove any intact tissue chunks.

[00288] The cells are washed using the swing bucket centrifuge set to 4°C, and the tube containing the cells is spun down at 500g for 5 minutes. The supernatant is aspirated and the cells are resuspended in 1 mL of PBS + 0.04% BSA. The wash is repeated two times. The final suspension is completed in a volume and buffer necessary and readied for analysis in a single cell assay.

[00289] The labeled cells are then processed in a droplet based single cell assay. Labeled cells are separated into individual droplets (FIG. 6) comprising the labeled cell and a gel bead. The labeled cell comprises the binding moieties, including a mixture of antibodies that bind to MHC Class I, CD45, and about 200 cell surface target proteins conjugated to a nucleic acid molecule comprising a linker and a capture sequence. The gel bead comprises nucleic acid molecules that comprise a primer binding site, a droplet barcode sequence, a unique molecular identifier (UMI) and a capture sequence or poly(dT) sequence. In the droplets (FIGs. 6B-6D), the nucleic acid molecules from the gel bead hybridize to a complementary sequence from the labeled cell. For example, the poly(dT) sequences from the gel bead hybridizes to the poly(A) sequences in the nucleic acid molecule comprising the spatial barcode (FIG. 6B), to the poly(A) tails of mRNA sequences from the cell (FIG. 6C), or to the poly(A) sequence in the nucleic acid molecule comprising the antibody barcode. In the droplets, reverse transcriptase (RT) transcribes nucleic acid molecules that comprise the droplet barcode and the spatial barcode, and the droplet barcode and the antibody barcode. The RT also transcribes nucleic acid molecules that comprise the droplet barcode and gene transcript.

[00290] The nucleic acid molecules transcribed by RT are amplified using a high-fidelity PCR with dual index primers. The amplicons are prepared for sequencing using a library preparation kit. The library is run on short-read sequencing machine and the sequences of each molecule are determined. Some sequences include a droplet barcode and a spatial barcode; these molecules determine which spatial barcode corresponds with which droplet. To determine which transcripts correspond with a cell in a particular location, the molecules with a droplet barcode are matched to the spatial barcode that is matched on the dual barcoded molecules. To determine which proteins are expressed in which cells, the molecules with a droplet barcode are matched to the spatial barcode that is matched to the droplet code on the nucleic acid molecule that encodes the antibody barcode. The transcriptome, proteome, and spatial location is then determined for the cells in the tissue.

Example 5. Spatially Analyzing Transcriptomes, Proteomes, and Epigenomes

[00291] To determine the location of cells in a tissue, a microwell array is provided to include binding moieties coupled to a spatial barcode (FIG. 7). In this example, each well of the microwell array includes a mixture of antibody binding moieties, each of which is conjugated to a spatial barcode corresponding to the array feature or well (FIG. 7). More specifically, each well of the microwell array includes an antibody conjugated to a first nucleic acid molecule that comprises a universal capture sequence, an antibody barcode sequence, and a first primer binding sequence (FIG. 7). The first nucleic acid molecule is hybridized to a second nucleic acid molecule that comprises the reverse complement of the universal capture sequence, a second primer binding sequence, a spatial barcode sequence, and a poly(A) sequence (FIG. 7). The microwell also comprises a nucleic acid molecule that comprises a mosaic end and a capture sequence that is configured to hybridize to Tn5 transposase (FIG. 7). The antibodies in a single micro well include a mixture of antibodies against target proteins, including MHC Class 1, CD45, CD3 and CD27, each coupled to a barcode corresponding to the same microwell. The antibody -barcode complex and transposase are printed in dry form into the microwell.

[00292] For example, the tissue comprises liver slices harvested from a subject, sectioned, frozen, and placed onto the microwell array over dry ice using forceps. In this example, a second array is introduced on the opposite side of the liver, and the liver will be sandwiched between the two arrays. Pressure is applied to the second array to seal the tissue in the array. The fluid from the tissue resuspends the dried antibody-barcode complexes immobilized in the micro wells. Once in solution, the antibodies bind the array of cell surface proteins on cells expressing the target protein. Once sealed, the array will be placed in a swing bucket centrifuge set to 50 RCF for 1 minute at 4°C. After centrifugation, the array will be incubated for 30 to 45 minutes at 37 °C to 42°C with agitation to dissociate the liver tissue.

[00293] The top array will then be removed and set aside face up so any liquid/tissue adhered is uncontaminated. The microwell array chip is held nearly perpendicular over the reagent reservoir so that liquid can drip down into the reservoir. Using a 200 pL pipette, 200 pL of the cell extraction buffer is withdrawn from the reservoir and “shot” into the wells of the chip to extract out tissue and cells, resuspending them and dripping them into the reagent reservoir. The buffer is introduced nearly perpendicular relative to the microwell array surface. The rinse is repeated several times, reusing the same buffer in the reservoir.

[00294] The process is repeated with the top array, introducing buffer onto the surface of the slide (or gasket) to resuspend tissue and cells that have loosely adhered and allowing them to drip into the reagent reservoir. All the cells and tissue are to be now present in the reagent reservoir. The mix is slowly pipetted lOx to mechanically break up larger tissue chunks and release cells. Using the same pipette, the cells are strained through a 70 pm cell strainer to remove any intact tissue chunks.

[00295] The cells are washed using the swing bucket centrifuge set to 4°C, and the tube containing the cells is spun down at 500g for 5 minutes. The supernatant is aspirated and the cells are resuspended in 1 mL of PBS + 0.04% BSA. The wash is repeated two times. The final suspension is completed in a volume and buffer necessary and readied for analysis in a single cell assay. [00296] The labeled cells are then processed in a droplet based single cell assay. Labeled cells from are separated into individual droplets (FIG. 7) comprising the labeled cell and a gel bead. The labeled cell comprises the binding moieties, including a mixture of antibodies that bind to MHC Class I, CD45, and about 200 cell surface target proteins conjugated to a nucleic acid molecule comprising a linker and a capture sequence. The gel bead comprises nucleic acid molecules that comprise a primer binding site, a droplet barcode sequence, a unique molecular identifier (UMI) and a capture sequence or poly(dT) sequence. In the droplets, the nucleic acid molecules from the gel bead hybridize to a complementary sequence from the labeled cell. For example, the poly(dT) sequences from the gel bead hybridize to the poly(A) sequences in the nucleic acid molecule comprising the spatial barcode, to the poly(A) tails of mRNA sequences from the cell, or to the poly(A) sequence in the nucleic acid molecule comprising the antibody barcode. The nucleic acid molecules configured to hybridize to Tn5 transposases recognize and isolate accessible chromatin. In the droplets, reverse transcriptase (RT) transcribes nucleic acid molecules that comprise the droplet barcode and the spatial barcode, and the droplet barcode and the antibody barcode. The RT also transcribes nucleic acid molecules that comprise the droplet barcode and gene transcript. An additional transcription reaction can also transcribe the nucleic acid molecules that comprise a mosaic end, adaptor, and sequences that correspond to accessible chromatin.

[00297] The nucleic molecules transcribed by RT are amplified using a high-fidelity PCR with dual index primers. The amplicons are prepared for sequencing using a library preparation kit. The library is run on short-read sequencing machine and the sequences of each molecule are determined. Some sequences include a droplet barcode and a spatial barcode; these molecules determine which spatial barcode corresponds with which droplet. To determine which transcripts correspond with a cell in a particular location, the molecules with a droplet barcode are matched to the spatial barcode that is matched on the dual barcoded molecules. To determine which proteins are expressed in which cells, the molecules with a droplet barcode are matched to the spatial barcode that is matched to the droplet code on the nucleic acid molecule that encodes the antibody barcode. The transcriptome, proteome, and spatial location is then determined for the cells in the tissue. To determine which chromatin is accessible in which cells, the mosaic ends and adaptors are sequenced. Example 6: unified single-cell and spatial analysis

[00298] In this experiment and as shown in FIG. 9, a mouse liver was sectioned to 300um slices and placed on a high density microarray (HDMA) for spatial labeling. In the left panel, cells are located based on spatial barcodes (top) compared to an image taken during tissue placement on the HDMA (bottom). Concordance of the top and bottom images illustrates the accuracy of spatial label and robustness of the assay. Colors in the top image denote different cell types, see the middle panel for cell identification. The middle panel shows a UMAP of cells identified by their transcriptomic profiling in a mouse liver tumor model. Identified cell types include abundant hepatocytes and macrophages, MC38 cancer cells as well as rare cell types such as cholangiocytes. Multiomics data (transcriptome and proteome) for individual cells are profiled and mapped according to their spatial locations.

[00299] A demonstration of how cells may be labeled with fluorescently tagged non-nucleotidyl moieties can be found in FIG. 12.

[00300] In FIG. 13, a tissue slice was labeled as described in Fig. 12 and the cells were dissociated. The resulting single cell suspension was analyzed using FACS. As the spatial reagents label the tissue layer that is in contact with the reagents, some cells are not labeled as shown in the lower left comer of the FACS plot. Labeled cells with dye 1 and dye 2 exhibit distinct color on the FACS plot. The result demonstrates successful spatial labeling and the labels are retained through tissue dissociation.

[00301] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be constmed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the ail without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for labeling cells or a subcellular compartment thereof, comprising:

(a) providing an array comprising a plurality of features each comprising one or more nucleic acid barcode molecules that comprise:

(i) a non-nucleotidyl moiety that binds to cells or a subcellular compartment thereof, and

(ii) a spatial barcode sequence that alone or in combination with one or more other spatial barcode sequences in a feature identifies a location of the feature on the array;

(b) contacting a tissue sample with the array and, while the tissue sample is contacted with the array, allowing the nucleic acid barcode molecules to couple to the cells or a subcellular compartment thereof in the tissue sample;

(c) removing the tissue sample from the array; and

(d) disassociating the cells or subcellular compartments thereof in the tissue sample after it has been removed from the array to produce a suspension comprising single cells or subcellular compartments that are labeled with one or more spatial barcode sequences that identify a location of a feature on the array.

2. The method of claim 1, further comprising:

(e) making a single-cell sequencing library from the suspension, a subset of cells thereof, or a subcellular compartment of either;

(f) sequencing the single-cell sequencing library to produce sequence reads;

(g) identifying one or more spatial barcode sequences in the sequence reads, and

(h) mapping the location of a cell or subcellular compartment to a feature of the array using the one or more spatial barcode sequences.

3. The method of claim 2, wherein: in (e) the single-cell sequencing library is an omics library that comprises:

(i) polynucleotides that comprise an analyte -related nucleic acid linked to a cell-specific barcode; and

(ii) polynucleotides that comprise a spatial barcode linked to a cell-specific barcode; the sequencing step of (f) produces omics data; and the method further comprises mapping the omics data for an individual cell or cellular compartment to a feature of the array using the one or more spatial barcodes for the individual cell or cellular compartment.

4. The method of claim 3, further comprising: reconstructing an image of the tissue sample using the mapped omics data.

5. The method of claim 1, wherein the non-nucleotidyl moiety of (a)(i) is: a binding agent that recognizes a protein, or a lipid that integrates into a membrane.

6. The method of claim 5, wherein the non-nucleotidyl moiety is an antibody.

7. The method of claim 5, wherein the non-nucleotidyl moiety is a fatty acid or cholesterol.

8. The method of claim 1, wherein a nucleic acid barcode molecule comprises:

(i) a first oligonucleotide that comprises a capture sequence and the non-nucleotidyl moiety;

(ii) a second oligonucleotide comprising a sequence that is complementary to the capture sequence of the first oligonucleotide, and the spatial barcode.

9. The method of claim 8, wherein the second oligonucleotide further comprises one or more universal processing sequences.

10. The method of claim 1, wherein the array is an array of microwells and each feature is a microwell.

11. The method of claim 1 , wherein the array is planar and the nucleic acid barcode molecules are on a planar surface.

12. The method of any one of claims 1-11, further comprising enriching for cells or a subcellular compartment from the suspension.

13. The method of any one of claims 1-12, wherein said tissue sample comprises a tissue section, a cell monolayer or an organoid and the tissue sample is fresh, frozen, fixed and/or permeabilized.

14. The method of any one of claims 2-13, wherein the single-cell sequencing library of (e) comprises cDNA, genomic DNA, probe sequences or ligation products containing the same, or barcodes that identify proteins or antibodies.

15. The method of any one of claims 1-14, wherein the non-nucleotidyl moiety binds to the surface of cells and wherein the suspension produced in (d) comprises single cells.

16. The method of any one of claims 1-14, wherein the non-nucleotidyl moiety binds to a subcellular compartment, and wherein the tissue sample is optionally fixed and/or permeabilized prior to step (b) to allow the non-nucleotidyl moiety into the cells, and wherein the suspension produced in (d) comprises single cells or subcellular compartments.

17. The method of claim 16, wherein subcellular compartments are nuclei.

18. The method of any one of claims 1-17, wherein the nucleic acid barcode molecule of (a) comprises a fluorescent moiety, and wherein the method comprises detecting the fluorescent moiety in the tissue sample, between steps (c) and (d).

19. The method of any one of claims 1-18, wherein, in (b), the tissue sample is sandwiched between two of the arrays, thereby allowing the nucleic acid barcode molecules from both arrays to couple to the cells or subcellular compartments thereof and, in step (c) the tissue sample is removed from both arrays.

20. The method of any one of claims 1-19, wherein, in (a), the nucleic acid barcode molecules are in solution.

21. The method of any one of claims 1-19, wherein the nucleic acid barcode molecules of (a) are in dried form, and they become hydrated upon contact with the tissue sample in step (b).

22. The method of any one of claims 1-21, wherein the nucleic acid barcode molecules of (a) are tethered to the array and step (b) comprises releasing the nucleic acid barcode molecules from the array.

23. A kit comprising an array of features, wherein a plurality of the features each comprise one or more nucleic acid barcode molecules that comprise:

(a) a non-nucleotidyl moiety that binds to cells or a subcellular compartment thereof, and

(b) a spatial barcode sequence that alone or in combination with one or more other spatial barcode sequences in a feature identifies a location of the feature on the array.

24. The kit of claim 23, wherein a nucleic acid barcode molecule comprises: (i) a first oligonucleotide that comprises a capture sequence and the non-nucleotidyl moiety; and

(ii) a second oligonucleotide comprising a sequence that is complementary to the capture sequence of the first oligonucleotide and the spatial barcode sequence.

25. The kit of claim 24, wherein the first oligonucleotide of (i) is the same in all features of the plurality of features.

26. The kit of claim 24 or 25, wherein the spatial barcode sequences vary from feature to feature in the plurality of features.

27. The kit of claim 24 or 25, wherein the second oligonucleotide further comprises one or more universal processing sequences, wherein the one or more universal processing sequences are the same in all features of the plurality of features.

28. The kit of any one of claims 23-27, wherein the plurality of features comprises at least 100 of the features.

29. The kit of any one of claims 23-28, wherein the features are wells.

30. The kit of any one of claims 23-28, wherein the array is planar and the nucleic acid barcode molecules are on a planar surface.

31. The kit of any one of claims 23-30, wherein the nucleic acid barcode molecules are in dry form.

32. The kit of claim 23 or 24, wherein the non-nucleotidyl moiety is a lipid that inserts into biological membranes.

33. The kit of claim 23 or 24, wherein the non-nucleotidyl moiety is an antibody.

34. The kit of claim 23 or 24, wherein the non-nucleotidyl moiety binds to the surface of cells.

35. The kit of any one of claims 23-33, wherein the non-nucleotidyl moiety binds to a subcellular compartment.

36. The kit of claim 35, wherein the non-nucleotidyl moiety binds to nuclei.