Bead-based single-cell secretome analysis

Abstract

Disclosed herein are systems, methods, compositions, and kits for measuring secreted analytes from cells, including those capable of simultaneously determining the secretory activity and protein and / or gene expression of a single cell. The disclosure herein includes a first solid support comprising a plurality of capture reagents capable of specifically binding to at least one of a plurality of secreted analytes secreted by a single cell. The disclosure herein also includes a secreted analyte binding reagent capable of specifically binding to the secreted analyte to which the capture reagent is bound. The secreted analyte binding reagent may comprise a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 421,512, filed November 1, 2022, the contents of which are incorporated herein by reference in their entirety for all purposes. The present disclosure relates generally to the field of molecular biology, for example, determining the secreted analyte profile of cells using molecular barcoding. [Background technology]

[0002] Current technology allows for the measurement of gene expression in single cells in a massively parallel fashion (e.g., over 10,000 cells) by attaching cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from individual cells, each of which colocalizes with a barcoded reagent bead in a compartment. Gene expression can affect protein expression and the secretion of molecules. Protein-protein interactions can affect gene expression, protein expression, and the secretion of molecules by cells. Cytokines and other molecules released by cells are of great interest to immunologists and other cell biologists. Traditional methods for detecting and measuring secreted proteins typically measure them at the bulk (rather than the single-cell level). For example, currently available methods include bead-based assays and ELISAs for testing secreted analytes in bulk. Therefore, single-cell quantification and cell phenotyping are lacking in the data. As with the comparison of flow cytometry and traditional Western blots, there is tremendous value in testing individual cells from a heterogeneous mixture of cells. There is an increasing need to correlate specific secretory activity with complex cellular phenotypes. Currently available methods for detecting secreted proteins are limited in the number of proteins that can be detected by the number of fluorescent markers that can be used for microscopy or flow cytometry analysis. Furthermore, such methods are less quantitative than desired due to the limited measurement of differences in fluorescence intensity. There is a need for systems and methods that can quantitatively analyze the copy number of secreted analytes secreted by single cells. There is a need for systems and methods that can quantitatively analyze the copy number of secreted analytes secreted by single cells, while simultaneously measuring protein expression and / or gene expression. Summary of the Invention

[0003] In some embodiments, a method is provided for measuring the copy number of a secreted analyte secreted by a single cell, the method comprising the steps of: distributing a plurality of first solid supports and one or more single cells into a plurality of compartments, each compartment comprising one or more first solid supports of a first plurality of first solid supports and one or more single cells, wherein the one or more single cells are capable of secreting a plurality of secreted analytes, each first solid support comprising a plurality of capture reagents capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell; and contacting the one or more first solid supports with a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte to which the capture reagent is bound, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent. contacting a plurality of oligonucleotide barcodes with secreted analyte-binding reagent-specific oligonucleotides for hybridization, wherein the oligonucleotide barcodes each comprise a first molecular label; extending the plurality of oligonucleotide barcodes hybridized to the secreted analyte-binding reagent-specific oligonucleotides to produce a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each barcode comprising a sequence complementary to at least a portion of a unique analyte identifier sequence and the first molecular label; and obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one secreted analyte secreted by each of the one or more single cells.

[0004] In some embodiments, methods are provided for determining the copy number of a secreted analyte secreted by a single cell and the copy number of a nucleic acid target in a single cell.The method includes the steps of: distributing a plurality of first solid supports and one or more single cells into a plurality of compartments, each compartment comprising one or more first solid supports of a first plurality of first solid supports and one or more single cells, wherein the one or more single cells comprise a copy of a nucleic acid target, and the one or more single cells are capable of secreting a plurality of secreted analytes, each first solid support comprising a plurality of capture reagents capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell; contacting the one or more first solid supports with a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte to which the capture reagent is bound, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent; and contacting a plurality of oligonucleotide barcodes for hybridization with the secreted analyte binding reagent-specific oligonucleotides and the copies of the nucleic acid target. extending the plurality of oligonucleotide barcodes hybridized to copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules, each comprising the first molecular label and a sequence complementary to at least a portion of the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the secreted analyte-binding reagent-specific oligonucleotides to generate a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each comprising the first molecular label and a sequence complementary to at least a portion of the unique analyte identifier sequence; obtaining sequence information of the plurality of barcoded nucleic acid molecules or products thereof to determine the copy number of the nucleic acid target in each of the one or more single cells; and obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one secreted analyte secreted by each of the one or more single cells.

[0005] In some embodiments, the one or more single cells comprise one or more single cells associated with a second solid support, and the method may include, prior to the distributing step, contacting the population of single cells with a plurality of second solid supports to generate one or more single cells associated with the second solid support, wherein the one or more single cells comprise a surface cell target, and each of the second solid supports comprises a plurality of isolation reagents, each of the plurality of isolation reagents being capable of specifically binding to the surface cell target, and the method may include removing a single cell of the population of single cells that is not associated with the second solid support. In some embodiments, the one or more single cells are one or more cell types of interest, and the cell types of interest may comprise surface cell targets to which the isolation reagents of the second solid support can bind.

[0006] The method may include, after contacting one or more first solid supports with a plurality of secreted analyte-binding reagents, removing one or more secreted analyte-binding reagents that are not in contact with one or more first solid supports of the plurality of secreted analyte-binding reagents. In some embodiments, removing one or more secreted analyte-binding reagents that are not in contact with one or more first solid supports comprises removing one or more secreted analyte-binding reagents that are not in contact with at least one of the secreted analytes bound by a respective capture reagent. In some embodiments, the first solid support and / or the second solid support comprises a magnetic material, optionally a ferromagnetic material. In some embodiments, the removing step may include applying a magnetic field to the plurality of compartments, and single cells associated with the second solid support and one or more first solid supports may be able to remain in the compartments when the magnetic field is applied. In some embodiments, the method comprises one or more incubation steps for a period of time, which may be about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, or about 240 minutes, and further, said incubation may occur after (i) contacting the single cell with a first solid support and / or (ii) contacting the one or more first solid supports with a plurality of secreted analyte binding reagents.

[0007] In some embodiments, a compartment of the plurality of compartments comprises about 2 to about 20 first solid supports, optionally about 8 first solid supports, which may be the same or different, and further, each first solid support may comprise a single type of capture reagent and / or may comprise different types of capture reagents. In some embodiments, the first solid support and / or the second solid support are less than about 15 μm. The one or more single cells may comprise T cells, B cells, tumor cells, bone marrow cells, blood cells, normal cells, fetal cells, maternal cells, or mixtures thereof. The method may include lysing the single cells in the compartment, wherein lysing the single cells may include heating the sample, contacting the sample with a detergent, altering the pH of the sample, or any combination thereof.

[0008] The at least one secreted analyte can include a lymphokine, an interleukin, a chemokine, or any combination thereof. For example, the secreted analyte can be a cytokine, a hormone, a molecular toxin, or any combination thereof. In some embodiments, the at least one secreted analyte includes a nerve growth factor, a liver growth factor, a fibroblast growth factor, a vascular endothelial growth factor, a platelet-derived growth factor, a transforming growth factor, an osteoinductive factor, an interferon, a colony-stimulating factor, or any combination thereof.

[0009] The secreted analyte binding reagent and the capture reagent may be capable of binding to distinct epitopes of the same secreted analyte. In some embodiments, one or more of the secreted analyte binding reagent, the capture reagent, and the isolation reagent comprise an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a monoclonal antibody. In some embodiments, the antibody or fragment thereof comprises a Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single domain antibody (sdAb), single chain comprising complementary scFvs (tandem scFvs) or bispecific tandem scFvs, Fv constructs, disulfide-linked Fv, dual variable domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, affibody, affilin, affitin, affimer, alphabody, anticalin, avimer, DARPin, Fynomer, Kunitz domain peptide, monobody, or any combination thereof. In some embodiments, the capture reagent and / or isolation reagent are conjugated to the first solid support and / or the second solid support by a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilic substitution reaction, a non-aldol carbonyl reaction, a carbon-carbon multiple bond addition, an oxidation reaction, a click reaction, or any combination thereof.

[0010] Surface cellular targets can include carbohydrates, lipids, proteins, extracellular proteins, cell surface proteins, cell markers, B cell receptors, T cell receptors, major histocompatibility complexes, tumor antigens, receptors, intracellular proteins, or any combination thereof. For example, surface cellular targets can include carbohydrates, lipids, proteins, or any combination thereof.

[0011] The plurality of oligonucleotide barcodes may be associated with a third solid support, and a compartment of the plurality of compartments comprises a single third solid support. In some embodiments, the compartment is a well or a droplet. In some embodiments, each oligonucleotide barcode comprises a first universal sequence. In some embodiments, the oligonucleotide barcode comprises a target binding region comprising a capture sequence. In some embodiments, the target binding region comprises a poly(dT) region. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide comprises a sequence complementary to a capture sequence configured to capture the secreted analyte-binding reagent-specific oligonucleotide. In some embodiments, the sequence complementary to the capture sequence comprises a poly(dA) region. In some embodiments, the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides comprises a complement of the first universal sequence. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide comprises a second universal sequence. In some embodiments, obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof comprises amplifying the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a second universal sequence or its complement to produce a plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides; and obtaining sequencing data of the plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof.

[0012] The secreted analyte-binding reagent-specific oligonucleotides may comprise a second molecular label. In some embodiments, at least 10 of the plurality of secreted analyte-binding reagent-specific oligonucleotides comprise different second molecular label sequences. In some embodiments, the second molecular label sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different, and the unique analyte identifier sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are identical. In some embodiments, the second molecular label sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different, and the unique analyte identifier sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different. In some embodiments, the number of unique first molecular label sequences associated with unique factor identifier sequences for secreted analyte-binding reagents capable of specifically binding to the at least one secreted analyte in the sequencing data indicates the copy number of the at least one secreted analyte secreted by each of the one or more single cells. In some embodiments, the number of unique second molecular label sequences associated with unique factor identifier sequences for secreted analyte-binding reagents capable of specifically binding to the at least one secreted analyte in the sequencing data indicates the copy number of the at least one secreted analyte secreted by each of the one or more single cells. The method may include determining the copy number of the at least one secreted analyte secreted by each of the one or more single cells based on the number of first molecular labels and / or second molecular labels having distinct sequences associated with a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof.

[0013] In some embodiments, the method comprises determining the copy number of at least one secreted analyte secreted by each of the one or more single cells based on the number of first molecular labels and / or second molecular labels having distinct sequences associated with the plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof. In some embodiments, obtaining the sequence information comprises attaching sequencing adaptors to the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof. The secreted analyte-binding reagent-specific oligonucleotide can include an alignment sequence adjacent to the poly(dA) region. The alignment sequence can be one or more nucleotides in length, or two or more nucleotides in length. For example, the alignment sequence can (a) include guanine, cytosine, thymine, uracil, or a combination thereof; (b) include poly(dT), poly(dG), poly(dC), poly(dU), or a combination thereof; and / or (c) be 5' to the poly(dA) region.

[0014] In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide is associated with the secreted analyte-binding reagent through a linker. In some embodiments, the linker comprises a carbon chain. The carbon chain can comprise 2 to 30 carbons (e.g., 12 carbons). In some embodiments, the linker comprises a 5' amino modifier C12 (5AmMC12) or a derivative thereof. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide is configured to be detachable from the secreted analyte-binding reagent. For example, the method can include dissociating the secreted analyte-binding reagent-specific oligonucleotide from the secreted analyte-binding reagent. In some embodiments, determining the copy number of the nucleic acid target in each of the one or more single cells comprises determining the copy number of the nucleic acid target in each of the one or more single cells based on the number of first molecular labels having distinct sequences, their complements, or combinations thereof, associated with the plurality of barcoded nucleic acid molecules or products thereof.

[0015] In some embodiments, the method includes contacting random primers with a plurality of barcoded nucleic acid molecules, each of the random primers comprising a third universal sequence or its complement, and extending the random primers hybridized to the plurality of barcoded nucleic acid molecules to generate a plurality of extension products. In some embodiments, the method includes amplifying the plurality of extension products using a primer capable of hybridizing to the first universal sequence or its complement and a primer capable of hybridizing to the third universal sequence or its complement, thereby generating a first plurality of barcoded amplicons. In some embodiments, amplifying the plurality of extension products includes adding sequences of sequencing primer and / or sequencing adapter binding sites, their complements, and / or portions thereof to the plurality of extension products. In some embodiments, the method includes determining the copy number of the nucleic acid target in each of one or more single cells based on the number of first molecular labels having distinct sequences associated with the first plurality of barcoded amplicons or products thereof.

[0016] Determining the copy number of the nucleic acid target in each of the one or more single cells may include determining the number of each of the plurality of nucleic acid targets in each of the one or more single cells based on the number of first molecular labels having distinct sequences associated with barcoded amplicons among a first plurality of barcoded amplicons that include the sequences of each of the plurality of nucleic acid targets. In some embodiments, the sequence of each of the plurality of nucleic acid targets comprises a subsequence of each of the plurality of nucleic acid targets. In some embodiments, the sequence of a nucleic acid target in the first plurality of barcoded amplicons comprises a subsequence of a nucleic acid target. In some embodiments, the method includes amplifying a first plurality of barcoded amplicons using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a third universal sequence or its complement, thereby generating a second plurality of barcoded amplicons. In some embodiments, amplifying the first plurality of barcoded amplicons includes adding sequences of sequencing primers and / or sequencing adapter binding sites, their complements, and / or portions thereof to the first plurality of barcoded amplicons. In some embodiments, the method includes determining the copy number of a nucleic acid target in each of one or more single cells based on the number of first molecular labels having distinct sequences associated with the second plurality of barcoded amplicons or products thereof. In some embodiments, the first plurality of barcoded amplicons and / or the second plurality of barcoded amplicons comprise whole transcriptome amplification (WTA) products.

[0017] In some embodiments, the method includes synthesizing a third plurality of barcoded amplicons using the plurality of barcoded nucleic acid molecules as templates to generate a third plurality of barcoded amplicons. In some embodiments, synthesizing the third plurality of barcoded amplicons includes (1) PCR amplification of the plurality of barcoded nucleic acid molecules, (2) PCR amplification using a primer capable of hybridizing to the first universal sequence or its complement and a target-specific primer, or both. In some embodiments, the method includes obtaining sequence information of the third plurality of barcoded amplicons or their products. Obtaining sequence information may include attaching sequencing adapters to the third plurality of barcoded amplicons or their products. The method may include determining the copy number of the nucleic acid target in each of the one or more single cells based on the number of first molecular labels having distinct sequences associated with the third plurality of barcoded amplicons or their products.

[0018] The nucleic acid target may include a nucleic acid molecule, such as ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, sample-indexing oligonucleotide, cellular component-binding reagent-specific oligonucleotide, or any combination thereof. In some embodiments, extending the plurality of oligonucleotide barcodes includes extending the plurality of oligonucleotide barcodes using a reverse transcriptase and / or a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. In some embodiments, the DNA polymerase includes a Klenow fragment. In some embodiments, the reverse transcriptase includes a viral reverse transcriptase (e.g., murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase).

[0019] In some embodiments, the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence are the same. In some embodiments, the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence are different. In some embodiments, the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence comprise a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof. In some embodiments, the sequencing adapter comprises a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof. In some embodiments, the sequencing primer comprises a lead 1 sequencing primer, a lead 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof. In some embodiments, at least 10 of the plurality of oligonucleotide barcodes comprise different first molecular label sequences. In some embodiments, each of the plurality of oligonucleotide barcodes comprises a cell label. In some embodiments, each cell label of the plurality of oligonucleotide barcodes comprises at least six nucleotides. The oligonucleotide barcodes associated with the same third solid support may comprise the same cell label. In some embodiments, the oligonucleotide barcodes associated with different third solid supports comprise different cell labels.

[0020] The first solid support, the second solid support, and / or the third solid support may comprise a synthetic particle or a planar surface. In some embodiments, at least one of the plurality of oligonucleotide barcodes is immobilized or partially immobilized on a synthetic particle, or at least one of the plurality of oligonucleotide barcodes is encapsulated or partially encapsulated within a synthetic particle. The synthetic particle may be disintegrable. The synthetic particles may include beads, such as sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or combinations thereof; materials selected from polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and combinations thereof; or disintegrable hydrogel particles.

[0021] In some embodiments, each of the plurality of oligonucleotide barcodes comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and combinations thereof. In some embodiments, each of the plurality of isolation reagents comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof.

[0022] In some embodiments, each of the plurality of capture reagents comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof. The one or more single cells may contain multiple cellular component targets. The method may further include the steps of contacting multiple cellular component binding reagents with the one or more single cells, each of the multiple cellular component binding reagents comprising a cellular component binding reagent-specific oligonucleotide comprising a unique identifier sequence for the cellular component binding reagent, the cellular component binding reagent being capable of specifically binding to at least one of the multiple cellular component targets, contacting multiple oligonucleotide barcodes with the cellular component binding reagent-specific oligonucleotides for hybridization, each of the oligonucleotide barcodes comprising a molecular label and a first universal sequence, extending the multiple oligonucleotide barcodes hybridized to the cellular component binding reagent-specific oligonucleotides to generate multiple barcoded cellular component binding reagent-specific oligonucleotides, each of the multiple barcodes comprising a molecular label and a sequence complementary to at least a portion of the unique identifier sequence, and obtaining sequence information of the multiple barcoded cellular component binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one of the multiple cellular component targets in each of the one or more single cells. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises a sequence complementary to a capture sequence configured to capture the cellular component binding reagent-specific oligonucleotide, hi some embodiments, the sequence complementary to the capture sequence comprises a poly(dA) region.

[0023] The plurality of barcoded cellular component binding reagent-specific oligonucleotides may comprise a complement of the first universal sequence. In some embodiments, the cellular component binding reagent-specific oligonucleotides comprise a fourth universal sequence. In some embodiments, obtaining sequence information of the plurality of barcoded cellular component binding reagent-specific oligonucleotides or their products includes amplifying the plurality of barcoded cellular component binding reagent-specific oligonucleotides or their products using a primer capable of hybridizing to the first universal sequence or its complement and a primer capable of hybridizing to the fourth universal sequence or its complement to generate a plurality of amplified barcoded cellular component binding reagent-specific oligonucleotides, and obtaining sequencing data of the plurality of amplified barcoded cellular component binding reagent-specific oligonucleotides or their products. The obtaining sequence information may include binding a sequencing adapter to the plurality of barcoded cellular component binding reagent-specific oligonucleotides or their products.

[0024] The method may include, after contacting the plurality of cellular component binding reagents with one or more single cells, removing one or more cellular component binding reagents from the plurality of cellular component binding reagents that are not in contact with one or more single cells. In some embodiments, removing one or more cellular component binding reagents that are not in contact with one or more single cells comprises removing one or more cellular component binding reagents that are not in contact with at least one of the plurality of cellular component targets. In some embodiments, the cellular component target comprises an intracellular protein, a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B-cell receptor, a T-cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof. In some embodiments, the cellular component target comprises a housekeeping protein, and detection of the housekeeping protein indicates the presence of a single cell in the compartment.

[0025] Some embodiments provide compositions (e.g., kits). In some embodiments, the composition includes a plurality of first solid supports comprising a plurality of capture reagents capable of specifically binding to at least one of a plurality of secreted analytes secreted by a single cell, and a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte bound by the capture reagent, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent. In some embodiments, the secreted analyte binding reagent and the capture reagent are capable of binding to distinct epitopes of the same secreted analyte. The composition may include a plurality of second solid supports comprising isolated reagents capable of specifically binding to a surface cellular target. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotides comprise a second molecular beacon sequence (e.g., 2-20 nucleotides in length). In some embodiments, the second molecular beacon sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different and the unique analyte identifier sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are identical. In some embodiments, the second molecular beacon sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different and the unique analyte identifier sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different.

[0026] In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide comprises a second universal sequence. In some embodiments, the second universal sequence comprises a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof. In some embodiments, the sequencing adapter comprises a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof. In some embodiments, the sequencing primer comprises a lead 1 sequencing primer, a lead 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof.

[0027] In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide comprises a poly(dA) region. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide comprises an alignment sequence adjacent to the poly(dA) region. The alignment sequence can be one or more nucleotides in length, or two or more nucleotides in length. The alignment sequence (a) can comprise guanine, cytosine, thymine, uracil, or a combination thereof; (b) can comprise a poly(dT), poly(dG), poly(dC), poly(dU), or a combination thereof; and / or (c) can be 5' to the poly(dA) region.

[0028] The secreted analyte-binding reagent-specific oligonucleotide may be associated with the secreted analyte-binding reagent through a linker. In some embodiments, the linker comprises a carbon chain. The carbon chain may comprise 2 to 30 carbons (e.g., 12 carbons). In some embodiments, the linker comprises a 5' amino modifier C12 (5AmMC12) or a derivative thereof. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide is attached to the secreted analyte-binding reagent. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide is covalently attached to the secreted analyte-binding reagent. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide is non-covalently attached to the secreted analyte-binding reagent. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide is conjugated to the secreted analyte-binding reagent. In some embodiments, the secreted analyte-binding reagent-specific oligonucleotide is conjugated to the secreted analyte-binding reagent through a chemical group such as a group cleavable by UV light, streptavidin, biotin, an amine, or a combination thereof.

[0029] The secreted analyte may include a lymphokine, an interleukin, a chemokine, or any combination thereof. For example, the secreted analyte may include a cytokine, a hormone, a molecular toxin, or any combination thereof. In some embodiments, the secreted analyte includes a nerve growth factor, a liver growth factor, a fibroblast growth factor, a vascular endothelial growth factor, a platelet-derived growth factor, a transforming growth factor, an osteoinductive factor, an interferon, a colony-stimulating factor, or any combination thereof.

[0030] The secreted analyte binding reagent and the capture reagent may be capable of binding to distinct epitopes on the same secreted analyte. In some embodiments, one or more of the secreted analyte binding reagent, capture reagent, and isolation reagent comprise an antibody or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a monoclonal antibody. In some embodiments, the antibody or fragment thereof comprises a Fab, Fab', F(ab')2, Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single domain antibody (sdAb), single chain including complementary scFv (tandem scFv) or bispecific tandem scFv, Fv construct, disulfide-linked Fv, dual variable domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, affibody, affilin, affitin, affimer, alphabody, anticalin, avimer, DARPin, phinomer, Kunitz domain peptide, monobody, or any combination thereof. In some embodiments, the capture reagent and / or isolation reagent are conjugated to the first solid support and / or the second solid support by a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilic substitution reaction, a non-aldol carbonyl reaction, a carbon-carbon multiple bond addition, an oxidation reaction, a click reaction, or any combination thereof.

[0031] In some embodiments, the surface cell target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof. In some embodiments, the composition comprises a DNA polymerase (e.g., a Klenow fragment) that lacks at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. In some embodiments, the composition comprises a reverse transcriptase, such as a viral reverse transcriptase. The composition may comprise a buffer, a cartridge, or both.

[0032] The composition may include a plurality of oligonucleotide barcodes, each of which comprises a target binding region. The target binding region may comprise a poly(dA) region, a poly(dT) region, a random sequence, a gene-specific sequence, or any combination thereof. In some embodiments, the plurality of oligonucleotide barcodes each comprise a molecular label. The molecular label may comprise at least six nucleotides. In some embodiments, at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences. The plurality of oligonucleotide barcodes may be associated with a third solid support. In some embodiments, the plurality of oligonucleotide barcodes each comprise a cell label. In some embodiments, the oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with the same third solid support comprise the same cell label. In some embodiments, the oligonucleotide barcodes of the plurality of oligonucleotide barcodes associated with different third solid supports comprise different cell labels.

[0033] In some embodiments, the first solid support, the second solid support, and / or the third solid support comprise synthetic particles or planar surfaces. At least one of the plurality of oligonucleotide barcodes may be immobilized or partially immobilized on a synthetic particle, or at least one of the plurality of oligonucleotide barcodes may be encapsulated or partially encapsulated within a synthetic particle. The synthetic particle may be collapsible, for example, a collapsible hydrogel particle. In some embodiments, each of the plurality of oligonucleotide barcodes comprises a linker functional group, and the synthetic particle comprises a solid support functional group, wherein the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and combinations thereof.

[0034] In some embodiments, each of the plurality of isolation reagents comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof. In some embodiments, each of the plurality of capture reagents comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof. [Brief explanation of the drawings]

[0035] [Figure 1] 1 shows a non-limiting exemplary barcode. [Figure 2] 1 illustrates a non-limiting exemplary workflow for barcoding and electronic counting. [Figure 3] 1 is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of 3′-barcoded targets from multiple targets. [Figure 4A] FIG. 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the copy number of one or more secreted analytes secreted by a single cell. [Figure 4B] FIG. 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the copy number of one or more secreted analytes secreted by a single cell. [Figure 4C]FIG. 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the copy number of one or more secreted analytes secreted by a single cell. [Figure 4D] FIG. 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the copy number of one or more secreted analytes secreted by a single cell. [Figure 4E] FIG. 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the copy number of one or more secreted analytes secreted by a single cell. [Figure 4F] FIG. 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the copy number of one or more secreted analytes secreted by a single cell. [Figure 4G] FIG. 1 shows a schematic diagram of a non-limiting exemplary workflow for determining the copy number of one or more secreted analytes secreted by a single cell. [Figure 5] FIG. 1 shows a non-limiting exemplary design of a secreted analyte-binding reagent-specific oligonucleotide (antibody oligonucleotide as shown herein) associated with a secreted analyte-binding reagent (antibody as shown herein). [Figure 6]

[0023] Figure 1 shows data regarding the use of the solid supports provided herein in Rhapsody cartridges. Arrow #1 indicates cells stained with calcein (bright green). Arrow #2 indicates dragon green 0.5 μm beads. Arrow #3 indicates CBA beads (7.5 μm). Arrow #4 indicates 15 μm beads. [Figure 7] A non-limiting exemplary workflow schematic (top panel) and data (bottom panel) are shown for the feasibility of a single-cell secretome workflow on the BD Rhapsody™ system. [Figure 8A] A non-limiting exemplary workflow schematic (Figure 8A) and data (Figures 8B-8C) are shown for a novel flow cytometry-based method for validating single-cell secretome (scS) beads and detection antibodies. [Figure 8B]A non-limiting exemplary workflow schematic (Figure 8A) and data (Figures 8B-8C) are shown for a novel flow cytometry-based method for validating single-cell secretome (scS) beads and detection antibodies. [Figure 8C] A non-limiting exemplary workflow schematic (Figure 8A) and data (Figures 8B-8C) are shown for a novel flow cytometry-based method for validating single-cell secretome (scS) beads and detection antibodies. [Figure 9A] A non-limiting exemplary workflow schematic (FIG. 9A) and data (FIG. 9B) are shown for detection of cytokine secretion using real-time PCR on a BD Rhapsody™ system. [Figure 9B] A non-limiting exemplary workflow schematic (FIG. 9A) and data (FIG. 9B) are shown for detection of cytokine secretion using real-time PCR on a BD Rhapsody™ system. DETAILED DESCRIPTION OF THE INVENTION

[0036] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless context dictates otherwise. The exemplary embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the drawings, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and make part of the disclosure herein. All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are incorporated by reference in their entirety for relevant art.

[0037] Quantifying a small number of nucleic acids, such as messenger ribonucleotide acid (mRNA) molecules, is clinically important, for example, to determine the genes expressed in cells at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can be very difficult, especially when the number of molecules is very small. One method for determining the absolute number of molecules in a sample is digital polymerase chain reaction (PCR). Ideally, PCR produces identical molecular copies in each cycle. However, PCR can have drawbacks because each molecule replicates with a stochastic probability, which varies depending on the PCR cycle and gene sequence, resulting in amplification bias and inaccurate gene expression measurements. Stochastic barcodes with unique molecular labels (also called molecular indexes (MIs)) can be used to count the number of molecules and correct for amplification bias. Stochastic barcoding, such as the Precise™ assay (Cellular Research, Inc., Palo Alto, CA) and the Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ), can correct for biases induced by the library preparation step by using molecular beacons (MLs) to label mRNA during PCR and reverse transcription (RT).

[0038] The Precise™ assay utilizes a non-depleting pool of stochastic barcodes with a large number of unique molecular tag sequences, e.g., 6561-65536, on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs in a sample during the RT step. The stochastic barcodes may contain universal PCR priming sites. During RT, target gene molecules react randomly with the stochastic barcodes. Each target molecule hybridizes to a stochastic barcode, resulting in the generation of a stochastically barcoded complementary ribonucleotide acid (cDNA) molecule. After labeling, the stochastically barcoded cDNA molecules from the microwells of a microwell plate may be pooled into a single tube for PCR amplification and sequencing. Raw sequencing data can be analyzed to obtain the number of reads, the number of stochastic barcodes with unique molecular tag sequences, and the number of mRNA molecules.

[0039] In some embodiments, a method is provided for measuring the copy number of a secreted analyte secreted by a single cell, the method comprising the steps of: distributing a plurality of first solid supports and one or more single cells into a plurality of compartments, each compartment comprising one or more first solid supports of a first plurality of first solid supports and one or more single cells, wherein the one or more single cells are capable of secreting a plurality of secreted analytes, each first solid support comprising a plurality of capture reagents capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell; and contacting the one or more first solid supports with a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte to which the capture reagent is bound, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent. contacting a plurality of oligonucleotide barcodes with secreted analyte-binding reagent-specific oligonucleotides for hybridization, wherein the oligonucleotide barcodes each comprise a first molecular label; extending the plurality of oligonucleotide barcodes hybridized to the secreted analyte-binding reagent-specific oligonucleotides to produce a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each barcode comprising a sequence complementary to at least a portion of a unique analyte identifier sequence and the first molecular label; and obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one secreted analyte secreted by each of the one or more single cells.

[0040] In some embodiments, methods are provided for determining the copy number of a secreted analyte secreted by a single cell and the copy number of a nucleic acid target in a single cell.The method includes the steps of: distributing a plurality of first solid supports and one or more single cells into a plurality of compartments, each compartment comprising one or more first solid supports of a first plurality of first solid supports and one or more single cells, wherein the one or more single cells comprise a copy of a nucleic acid target, and the one or more single cells are capable of secreting a plurality of secreted analytes, each first solid support comprising a plurality of capture reagents capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell; contacting the one or more first solid supports with a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte to which the capture reagent is bound, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent; and contacting a plurality of oligonucleotide barcodes for hybridization with the secreted analyte binding reagent-specific oligonucleotides and the copies of the nucleic acid target. extending the plurality of oligonucleotide barcodes hybridized to copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules, each comprising the first molecular label and a sequence complementary to at least a portion of the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the secreted analyte-binding reagent-specific oligonucleotides to generate a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each comprising the first molecular label and a sequence complementary to at least a portion of the unique analyte identifier sequence; obtaining sequence information of the plurality of barcoded nucleic acid molecules or products thereof to determine the copy number of the nucleic acid target in each of the one or more single cells; and obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one secreted analyte secreted by each of the one or more single cells.

[0041] In some embodiments, a composition (e.g., a kit) is provided. In some embodiments, the composition comprises a plurality of first solid supports comprising a plurality of capture reagents capable of specifically binding to at least one of a plurality of secreted analytes secreted by a single cell, and a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte to which the capture reagent is bound, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent. In some embodiments, the secreted analyte binding reagent and the capture reagent are capable of binding to distinct epitopes of the same secreted analyte. The composition may comprise a plurality of second solid supports comprising isolated reagents capable of specifically binding to surface cellular targets.

[0042] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0043] As used herein, the term "adapter" may refer to a sequence for facilitating amplification or sequencing of an associated nucleic acid. The associated nucleic acid may include a target nucleic acid. The associated nucleic acid may include one or more of a spatial label, a target label, a sample label, an indexing label, or a barcode sequence (e.g., a molecular label). The adapter may be linear. The adapter may be a pre-adenylated adapter. The adapter may be double-stranded or single-stranded. One or more adapters may be positioned at the 5' or 3' end of a nucleic acid. When an adapter includes known sequences at the 5' and 3' ends, the known sequences may be the same or different sequences. The adapters positioned at the 5' and / or 3' ends of a polynucleotide may be capable of hybridizing to one or more oligonucleotides immobilized on a surface. In some embodiments, the adapter may include a universal sequence. A universal sequence may be a region of nucleotide sequence that is common to two or more nucleic acid molecules. The two or more nucleic acid molecules may also have regions of different sequences. Thus, for example, the 5' adapters can contain identical and / or universal nucleic acid sequences, and the 3' adapters can contain identical and / or universal sequences. The presence of a universal sequence in different members of a plurality of nucleic acid molecules can allow for the replication or amplification of multiple different sequences using a single universal primer that is complementary to the universal sequence. Similarly, at least one, two (e.g., a pair), or more universal sequences can be present in different members of a collection of nucleic acid molecules, allowing for the replication or amplification of multiple different sequences using at least one, two (e.g., a pair), or more single universal primers that are complementary to the universal sequence. Thus, a universal primer includes a sequence that can hybridize to such a universal sequence. A molecule having a target nucleic acid sequence can be modified to add universal adapters (e.g., non-target nucleic acid sequences) to one or both ends of the different target nucleic acid sequences.The one or more universal primers bound to the target nucleic acid may provide a site for hybridization of the universal primer. The one or more universal primers bound to the target nucleic acid may be the same or different from each other.

[0044] As used herein, the terms "associated" or "associated with" can mean that two or more species are identifiable as being located together at a time. Association can mean that two or more species are or were located in similar containers. Association can also be an informational association. For example, digital information about two or more species can be stored and used to determine that one or more of the species were located together at a time. Association can also be a physical association. In some embodiments, two or more associated species are "tethered," "bound," or "immobilized" to each other or to a common solid or semi-solid surface. Association can refer to covalent or non-covalent means for attaching a label to a solid or semi-solid support, such as a bead. Association can also be a covalent bond between a target and a label. Association can include hybridization between two molecules (e.g., a target molecule and a label).

[0045] As used herein, the term "complementary" may refer to the ability for precise pairing between two nucleotides. For example, if a nucleotide at a given position in a nucleic acid can hydrogen bond with a nucleotide in another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules may be "partial," in which only some of the nucleotides bind, or may be complete, in which total complementarity exists between the single-stranded molecules. A first nucleotide sequence may be referred to as the "complement" of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence may be referred to as the "reverse complement" of a second sequence if the first nucleotide sequence is complementary to a sequence that is the reverse of the second sequence (i.e., the order of the nucleotides is reversed). As used herein, a "complementary" sequence may refer to the "complement" or "reverse complement" of a sequence. It is understood from this disclosure that when a molecule is capable of hybridizing to another molecule, it may be complementary or partially complementary to the hybridizing molecule.

[0046] As used herein, the term "digital counting" can refer to a method for estimating the number of target molecules in a sample. Digital counting can include determining the number of unique labels associated with targets in a sample. This methodology, which can be probabilistic in nature, transforms the molecular counting problem into one of locating and identifying identical molecules into a series of yes / no digital questions regarding the detection of a predefined set of labels.

[0047] As used herein, the term "label" or "labels" can refer to a nucleic acid code associated with a target in a sample. The label can be, for example, a nucleic acid label. The label can be a wholly or partially amplifiable label. The label can be a wholly or partially sequenceable label. The label can be a portion of a naturally occurring nucleic acid that can be identified as distinct. The label can be a known sequence. The label can include a junction of a nucleic acid sequence, e.g., a junction of a naturally occurring and non-naturally occurring sequence. As used herein, the term "label" can be used interchangeably with the terms "index," "tag," or "label tag." The label can carry information. For example, in various embodiments, the label can be used to determine the identity of the sample, the source of the sample, the identity of the cell, and / or the target.

[0048] As used herein, the term "non-depletion reservoir" can refer to a pool of barcodes (e.g., stochastic barcodes) composed of many different labels. The non-depletion reservoir can contain many different barcodes so that when the non-depletion reservoir is associated with a pool of targets, each target is likely to associate with a unique barcode. The uniqueness of each labeled target molecule can be determined by random selection statistics and depends on the copy number of identical target molecules in the population compared to the diversity of the labels. The size of the resulting labeled target molecule can be determined by the stochastic nature of the barcoding process, and analysis of the number of detected barcodes then allows for calculation of the number of target molecules present in the original population or sample. If the ratio of the number of target molecules present to the number of unique barcodes is low, the labeled target molecule is highly unique (i.e., the probability that more than one target molecule will be labeled with a given label is very low).

[0049] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. A nucleic acid may comprise nucleotides. A nucleic acid may be exogenous or endogenous to a cell. A nucleic acid may exist in a cell-free environment. A nucleic acid may be a gene or a fragment thereof. A nucleic acid may be DNA. A nucleic acid may be RNA. A nucleic acid may comprise one or more analogs (e.g., modified backbones, sugars, or nucleobases). Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" can be used interchangeably.

[0050] Nucleic acids may contain one or more modifications (e.g., base modifications, backbone modifications) to result in nucleic acids with new or enhanced properties (e.g., improved stability). Nucleic acids may also contain nucleic acid affinity tags. A nucleoside may be a base-sugar combination. The base portion of a nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide may be a nucleoside further comprising a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleosides containing a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate group may covalently link adjacent nucleosides to one another to form a linear polymeric compound. The respective ends of this linear polymeric compound may then be further joined to form a circular compound, although linear compounds are generally preferred. In addition, linear compounds may have internal nucleotide base complementarity and therefore may fold in such a manner as to yield fully or partially double-stranded compounds. Within nucleic acids, the phosphate groups may commonly be referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone may be a 3' and 5' phosphodiester linkage.

[0051] The nucleic acids can contain modified backbones and / or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs, and those with reverse polarity, wherein one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.

[0052] Nucleic acids can contain polynucleotide backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH moieties.

[0053] Nucleic acids may include nucleic acid mimetics. The term "mimetics" is intended to include polynucleotides in which only the furanose ring, or both the furanose ring and the internucleotide linkage, are replaced with non-furanose groups; replacement of only the furanose ring may also be referred to as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety may be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid may be a peptide nucleic acid (PNA). In a PNA, the sugar backbone of a polynucleotide may be replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotides may be retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. The backbone in a PNA compound may contain two or more linked aminoethylglycine units, thereby providing the PNA with an amide-containing backbone. The heterocyclic base moiety may be directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. The nucleic acid may include a morpholino backbone structure. For example, the nucleic acid may include a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, phosphorodiamidates or other non-phosphodiester internucleoside linkages may replace the phosphodiester linkages.

[0054] Nucleic acids can contain linked morpholino units (e.g., morpholino nucleic acids) with heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds may have fewer undesirable interactions with intracellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds within the morpholino class can be attached using different linking groups. A further class of polynucleotide mimics can be called cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared and used to synthesize oligomeric compounds using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid chains can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to that of native complexes. Further modifications include locked nucleic acids (LNAs), in which a 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2) group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNAs and LNA analogs can exhibit very high duplex thermal stability with complementary nucleic acids (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility characteristics.

[0055] Nucleic acids can also include nucleobase (often simply referred to as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases can include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkyl derivatives of the pyrimidine base, 6-azouracil, cytosine ... Mention may be made of tosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps, such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidines (2H-pyrimido(4,5-b)indol-2-one), and pyridoindole cytidines (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0056] As used herein, the term "sample" can refer to a composition that contains a target. Samples suitable for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms. As used herein, the terms "sample collection device" or "device" may refer to a device capable of collecting a section of a sample and / or placing the section on a substrate. A sample device may refer to, for example, a fluorescence activated cell sorter (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and / or a microtome.

[0057] As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which multiple barcodes (e.g., stochastic barcodes) can be attached. A solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar structure composed of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) to which nucleic acids can be immobilized (e.g., covalently or non-covalently). A solid support may comprise discrete particles that may be spherical (e.g., microspheres) or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. A bead may be non-spherical in shape. A plurality of solid supports spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "bead."

[0058] As used herein, the term "stochastic barcode" may refer to a polynucleotide sequence comprising a label of the present disclosure. A stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. A stochastic barcode may be used to quantify a target within a sample. A stochastic barcode may be used to control errors that may occur after associating a label with a target. For example, a stochastic barcode may be used to evaluate amplification or sequencing errors. A stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target. As used herein, the term "gene-specific stochastic barcode" may refer to a polynucleotide sequence comprising a label and a gene-specific target-binding region. A stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. A stochastic barcode may be used to quantify a target within a sample. A stochastic barcode may be used to control errors that may occur after associating a label with a target. For example, a stochastic barcode may be used to evaluate amplification or sequencing errors. A stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target.

[0059] As used herein, the term "stochastic barcoding" can refer to random labeling (e.g., barcoding) of nucleic acids. Stochastic barcoding can utilize a Poisson recursion strategy to associate labels and quantify the labels associated with targets. As used herein, the term "stochastic barcoding" can be used interchangeably with "stochastic labeling." As used herein, the term "target" may refer to a composition that can be associated with a barcode (e.g., a stochastic barcode). Exemplary targets suitable for analysis by the methods, devices, and systems of the present disclosure include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, and the like. Targets may be single-stranded or double-stranded. In some embodiments, targets may be proteins, peptides, or polypeptides. In some embodiments, targets are lipids. As used herein, "target" may be used interchangeably with "species."

[0060] As used herein, the term "reverse transcriptase" can refer to a group of enzymes that have reverse transcriptase activity (i.e., that catalyze the synthesis of DNA from an RNA template). Generally, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptases, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retron reverse transcriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include the Lactococcus lactis LI.LtrB intron reverse transcriptase, the Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or the Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases include the numerous classes of non-retroviral reverse transcriptases (i.e., retrons, group II introns, and diversity-generating retroelements, among others).

[0061] The terms "universal adapter primer," "universal primer adapter," or "universal adapter sequence" are used interchangeably to refer to a nucleotide sequence that can hybridize to a barcode (e.g., a stochastic barcode) and be used to generate a gene-specific barcode. A universal adapter sequence can be, for example, a known sequence that is universal across all barcodes used in the methods of the present disclosure. For example, when multiple targets are labeled using the methods disclosed herein, each of the target-specific sequences can be ligated to the same universal adapter sequence. In some embodiments, two or more universal adapter sequences can be used in the methods disclosed herein. For example, when multiple targets are labeled using the methods disclosed herein, at least two of the target-specific sequences are ligated to different universal adapter sequences. The universal adapter primer and its complement can be included in two oligonucleotides, one of which contains the target-specific sequence and the other of which contains the barcode. For example, a universal adapter sequence can be part of an oligonucleotide that contains a target-specific sequence to generate a nucleotide sequence that is complementary to a target nucleic acid. A second oligonucleotide comprising the complement of the barcode and universal adapter sequence can hybridize to the nucleotide sequence to generate a target-specific barcode (target-specific stochastic barcode). In some embodiments, the universal adapter primer has a different sequence than the universal PCR primer used in the disclosed methods.

[0062] Barcode Barcoding, e.g., probabilistic barcoding, is described, for example, in Fu et al., Proc Natl Acad Sci USA, 2011 May 31, 108(22):9026-31; US2011 / 0160078; Fan et al., Science, 2015 February 6, 347(6222):1258367; US2015 / 0299784; and WO2015 / 031691, the contents of each of which, including any supplemental or additional information, are incorporated herein by reference in their entirety. In some embodiments, the barcodes disclosed herein may be probabilistic barcodes, which may be polynucleotide sequences that can be used to stochastically label (e.g., barcode, tag) targets. A barcode may be referred to as a stochastic barcode if the ratio of the number of distinct barcode sequences in the stochastic barcode to the number of occurrences of any of the targets to be labeled can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values, or can be approximately these values ​​or such numbers or ranges. The targets may be mRNA species that include mRNA molecules with identical or nearly identical sequences. A barcode may be referred to as a stochastic barcode if the ratio of the number of distinct barcode sequences of the stochastic barcode to the number of occurrences of any of the targets to be labeled is at least or at most 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1. The barcode sequences of a stochastic barcode may be referred to as molecular labels.

[0063] A barcode, e.g., a stochastic barcode, can include one or more labels. Exemplary labels can include a universal label, a cell label, a barcode sequence (e.g., a molecular label), a sample label, a plate label, a spatial label, and / or a pre-spatial label. FIG. 1 shows an exemplary barcode 104 having a spatial label. The barcode 104 can include a 5' amine that can link the barcode to a solid support 105. The barcode can include a universal label, a dimensional label, a spatial label, a cell label, and / or a molecular label. The order of different labels (including, but not limited to, the universal label, the dimensional label, the spatial label, the cell label, and the molecular label) within the barcode can vary. For example, as shown in FIG. 1, the universal label can be the 5'-most label and the molecular label can be the 3'-most label. The spatial label, the dimensional label, and the cell label can be in any order. In some embodiments, the universal label, the spatial label, the dimensional label, the cell label, and the molecular label are in any order. The barcode can include a target binding region. The target binding region can interact with a target (e.g., target nucleic acid, RNA, mRNA, DNA) in a sample. For example, the target binding region can include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. The labels of the barcode (e.g., universal label, dimensional label, spatial label, cellular label, and barcode sequence) can be spaced apart by, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides.

[0064] Labels, e.g., cellular labels, can include a set of unique nucleic acid subsequences of defined length, e.g., seven nucleotides each (equivalent to the number of bits used in some Hamming error-correcting codes), that can be designed to provide error-correcting capabilities. An error-correcting subsequence set including seven-nucleotide sequences can be designed so that any pairwise combination of sequences in the set exhibits a defined "genetic distance" (or number of mismatched bases); for example, an error-correcting subsequence set can be designed to exhibit a genetic distance of three nucleotides. In this case, consideration of the error-correcting sequences in a sequence dataset of a labeled target nucleic acid molecule (described in more detail below) can enable detection or correction of amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequences used to create the error-correcting code can vary, e.g., be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides, or a number or range between or about any two of these values. In some embodiments, nucleic acid subsequences of other lengths may be used to create error-correcting codes.

[0065] The barcode may include a target binding region. The target binding region may interact with a target in the sample. The target may be or include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNAs each including a poly(A) tail, or any combination thereof. In some embodiments, the multiple targets may include deoxyribonucleic acid (DNA).

[0066] In some embodiments, the target binding region may include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. One or more of the labels of the barcode (e.g., universal label, dimensional label, spatial label, cellular label, and barcode sequence (e.g., molecular label)) may be separated from another one or two of the remaining labels of the barcode by a spacer. The spacer may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, none of the labels of the barcode are separated by a spacer.

[0067] Universal Signage A barcode may include one or more universal labels. In some embodiments, the one or more universal labels may be the same for all barcodes in a set of barcodes bound to a given solid support. In some embodiments, the one or more universal labels may be the same for all barcodes bound to a plurality of beads. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a sequencing primer. The sequencing primer can be used to sequence barcodes that include a universal label. The sequencing primer (e.g., a universal sequencing primer) may include a sequencing primer associated with a high-throughput sequencing platform. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a PCR primer. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a sequencing primer and a PCR primer. The nucleic acid sequence of a universal label that can hybridize to a sequencing primer or a PCR primer may be referred to as a primer binding site. The universal label may include a sequence that can be used to initiate transcription of the barcode. A universal label can include a sequence that can be used to extend a barcode or a region within the barcode. A universal label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between or about any two of these values. For example, a universal label can include at least about 10 nucleotides. A universal label can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. In some embodiments, a cleavable linker or modified nucleotides can be part of the universal label sequence to allow the barcode to be cleaved from the support.

[0068] dimensional indicator A barcode may include one or more dimensional labels. In some embodiments, a dimensional label may include a nucleic acid sequence that provides information about the dimension in which labeling (e.g., stochastic labeling) occurred. For example, a dimensional label can provide information about the time a target was barcoded. A dimensional label may be associated with the time of barcoding (e.g., stochastic barcoding) in a sample. A dimensional label may be activated at the time of labeling. Different dimensional labels may be activated at different time points. A dimensional label provides information about the order in which a target, a group of targets, and / or a sample was barcoded. For example, a cell population may be barcoded in the G0 phase of the cell cycle. Cells may be pulsed again with a barcode (e.g., a stochastic barcode) in the G1 phase of the cell cycle. Cells may be pulsed again with a barcode in the S phase of the cell cycle, and so on. The barcode in each pulse (e.g., each stage of the cell cycle) may include a different dimensional label. In this way, the dimensional label provides information about which targets were labeled at which stage of the cell cycle. Dimensional labeling can examine many different biological time periods. Exemplary biological time periods can include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., a cell, a cell population) can be stochastically labeled before and / or after treatment with a drug and / or therapy. Changes in copy number of distinct targets can indicate the sample's response to the drug and / or therapy.

[0069] Dimensional labels may be activatable. Activatable dimensional labels can be activated at a specific time. Activatable labels can, for example, be continuously activated (e.g., not turned off). Activatable dimensional labels can, for example, be reversibly activatable (e.g., they can be turned on and turned off). Dimensional labels can be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Dimensional labels can be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, dimensional labels can be activated by fluorescence, light, chemical events (e.g., cleavage, ligation of another molecule, addition of a modification (e.g., pegylation, sumoylation, acetylation, deacetylation, demethylation), photochemical events (e.g., photocaging), and introduction of unnatural nucleotides.

[0070] In some embodiments, the dimension labels may be the same for all barcodes (e.g., stochastic barcodes) attached to a given solid support (e.g., a bead), but may be different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the barcodes on the same solid support may comprise the same dimension label. In some embodiments, at least 60% of the barcodes on the same solid support may comprise the same dimension label. In some embodiments, at least 95% of the barcodes on the same solid support may comprise the same dimension label. On multiple solid supports (e.g., beads), 6Many unique dimension label sequences, even more than 10, may be presented. Dimension labels can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Dimension labels can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. Dimension labels can comprise from about 5 to about 200 nucleotides. Dimension labels can comprise from about 10 to about 150 nucleotides. Dimension labels can comprise from about 20 to about 125 nucleotides in length.

[0071] spatial sign A barcode may include one or more spatial labels. In some embodiments, a spatial label may include a nucleic acid sequence that provides information about the spatial orientation of a target molecule associated with the barcode. A spatial label may be associated with a coordinate in a sample. The coordinate may be a fixed coordinate. For example, the coordinate may be fixed relative to a substrate. A spatial label may reference a two-dimensional or three-dimensional grid. The coordinate may be fixed relative to a landmark. The landmark may be identifiable in space. The landmark may be a structure that can be imaged. The landmark may be a biological structure, e.g., an anatomical landmark. The landmark may be a cellular landmark, e.g., an organelle. The landmark may be a non-natural landmark, e.g., an identifiable identifier, e.g., a color code, a barcode, a magnetic property, fluorescence, radioactivity, or a structure with a unique size or shape. A spatial label may be associated with a physical compartment (e.g., a well, a container, or a droplet). In some embodiments, multiple spatial labels are used together to encode one or more locations in space.

[0072] Spatial labels may be the same for all barcodes attached to a given solid support (e.g., beads), but may be different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or may be approximately these values ​​or such numbers or ranges. In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support may be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, at least 60% of the barcodes on the same solid support may contain the same spatial label. In some embodiments, at least 95% of the barcodes on the same solid support may contain the same spatial label.

[0073] On multiple solid supports (e.g., beads), 6 Many unique spatial marker sequences, even more than 10, may be presented. Spatial markers can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Spatial markers can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. Spatial markers can comprise between about 5 and about 200 nucleotides. Spatial markers can comprise between about 10 and about 150 nucleotides. Spatial markers can comprise between about 20 and about 125 nucleotides in length.

[0074] cell labeling A barcode (e.g., a stochastic barcode) may include one or more cell labels. In some embodiments, the cell label may include a nucleic acid sequence that provides information for determining which target nucleic acid originated from which cell. In some embodiments, the cell label is the same for all barcodes attached to a given solid support (e.g., a bead) but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be, or approximately, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be, or approximately, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least 60% of the barcodes on the same solid support may contain the same cell label. As another example, at least 95% of the barcodes on the same solid support may contain the same cell label. On multiple solid supports (e.g., beads), 6 Many unique cell marker sequences, even more than 10, may be presented. Cell markers can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Cell markers can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. For example, a cell marker can comprise between about 5 and about 200 nucleotides. As another example, a cell marker can comprise between about 10 and about 150 nucleotides. As yet another example, a cell marker can comprise between about 20 and about 125 nucleotides in length.

[0075] Barcode sequence The barcode may include one or more barcode sequences. In some embodiments, the barcode sequence may include a nucleic acid sequence that provides information about the specific type of target nucleic acid species hybridized to the barcode. The barcode sequence includes a nucleic acid sequence that provides a counter (e.g., provides a rough approximation) for the specific occurrence of the target nucleic acid species hybridized to the barcode (e.g., target binding region).

[0076] In some embodiments, a diverse set of barcode sequences is attached to a given solid support (e.g., a bead). 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 There may be at least 10, or a number or range between, or about, any two of these values, or such number or range of unique molecular label sequences. For example, the plurality of barcodes may include about 6561 barcode sequences having distinct sequences. As another example, the plurality of barcodes may include about 65536 barcode sequences having distinct sequences. In some embodiments, there may be at least or at most 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 There may be multiple unique barcode sequences. The unique molecular beacon sequences may be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular beacon sequences are partially or entirely encompassed by a particle (e.g., a hydrogel bead).

[0077] The length of the barcode may vary in different implementations. For example, the barcode may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, or may be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, in length. As another example, the barcode may be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0078] molecular label A barcode (e.g., a stochastic barcode) can include one or more molecular labels. A molecular label can include a barcode sequence. In some embodiments, a molecular label can include a nucleic acid sequence that provides information about the specific type of target nucleic acid species hybridized to the barcode. A molecular label includes a nucleic acid sequence that provides a counter for the specific occurrence of a target nucleic acid species hybridized to the barcode (e.g., a target binding region). In some embodiments, a diverse set of molecular labels is attached to a given solid support (e.g., beads). 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 or a number or range between any two of these values, or about 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9There may be at least 10 unique molecular label sequences, or a number or range between any two of these values. For example, the plurality of barcodes may include about 6561 molecular labels with distinct sequences. As another example, the plurality of barcodes may include about 65536 molecular labels with distinct sequences. In some embodiments, there may be at least or at most 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 There can be a number of unique molecular beacon sequences. A barcode with a unique molecular beacon sequence can be attached to a given solid support (e.g., a bead).

[0079] For barcoding using multiple stochastic barcodes (e.g., stochastic barcoding), the ratio of the number of distinct molecular label sequences to the number of occurrences of any of the targets can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values, or may be about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. The target may be an mRNA species that includes mRNA molecules with identical or nearly identical sequences. In some embodiments, the ratio of the number of different molecular beacon sequences to the number of occurrences of any of the targets is at least or at most 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

[0080] A molecular label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or a number or range of nucleotides approximately equal to or equal to these values ​​or such number or range. A molecular label can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0081] Target binding region The barcode may include one or more target binding regions, e.g., capture probes. In some embodiments, the target binding region may hybridize with a target of interest. In some embodiments, the target binding region may include a nucleic acid sequence that specifically hybridizes to a target (e.g., a target nucleic acid, target molecule, e.g., a cellular nucleic acid to be analyzed), e.g., a specific gene sequence. The target binding region may include, for example, a nucleic acid sequence that can bind (e.g., hybridize) to a specific position of a specific target nucleic acid. In some embodiments, the target binding region may include a nucleic acid sequence that is capable of specific hybridization to a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode can then be ligated to any nucleic acid molecule that includes a sequence complementary to the restriction site overhang.

[0082] In some embodiments, the target binding region may comprise a non-specific target nucleic acid sequence. A non-specific target nucleic acid sequence may refer to a sequence that can bind to multiple target nucleic acids independently of the specific sequence of the target nucleic acid. For example, the target binding region may comprise a random multimer sequence, a poly(dA) sequence, a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, or a combination thereof. For example, the target binding region may be an oligo(dT) sequence that hybridizes to a poly(A) tail on an mRNA molecule. The random multimer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or any longer multimer sequence of any length. In some embodiments, the target binding region is the same for all barcodes bound to a given bead. In some embodiments, the target binding regions of multiple barcodes bound to a given bead may comprise two or more different target binding sequences. The target binding region can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. The target binding region can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or more. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to generate a cDNA molecule with a poly(dC) tail. The barcode can include a target binding region with a poly(dG) tail. Upon base pairing between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues replication to the 5' end of the barcode. In doing so, the resulting cDNA molecules contain a barcode sequence (eg, a molecular tag) on ​​the 3' end of the cDNA molecule.

[0083] In some embodiments, the target binding region may comprise an oligo(dT) capable of hybridizing to an mRNA containing a polyadenylated end. The target binding region may be gene-specific. For example, the target binding region may be configured to hybridize to a specific region of the target. The target binding region may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or a number or range between any two of these values, or approximately these values ​​or such number or range, in length. The target binding region can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The target binding region can be about 5-30 nucleotides in length. When a barcode includes a gene-specific target binding region, the barcode may be referred to herein as a gene-specific barcode.

[0084] Orientation Characteristics A stochastic barcode (e.g., a stochastic barcode) may include one or more orientation properties that can be used to orient (e.g., align) the barcode. The barcode may include a moiety for isoelectric focusing. Different barcodes may include different isoelectric focusing points. When these barcodes are introduced into a sample, the sample may undergo isoelectric focusing to orient the barcodes in a known manner. In this manner, the orientation properties can be used to develop a known map of the barcodes in the sample. Exemplary orientation properties can include electrophoretic mobility (e.g., based on the size of the barcode), isoelectric point, spin, conductivity, and / or self-assembly. For example, a barcode with an orientation property for self-assembly may self-assemble into a specific orientation (e.g., a nucleic acid nanostructure) when activated.

[0085] affinity properties A barcode (e.g., a stochastic barcode) can include one or more affinity features. For example, a spatial label can include an affinity feature. Affinity features can include chemical and / or biological moieties that can facilitate binding of the barcode to another entity (e.g., a cellular receptor). For example, an affinity feature can include an antibody, e.g., an antibody specific to a particular moiety (e.g., a receptor) on a sample. In some embodiments, the antibody can direct the barcode to a particular cell type or molecule. Targets on and / or near a particular cell type or molecule can be labeled (e.g., stochastically labeled). In some embodiments, the affinity feature can provide spatial information in addition to the nucleotide sequence of the spatial label, as the antibody can direct the barcode to a specific location. The antibody can be a therapeutic antibody, e.g., a monoclonal or polyclonal antibody. The antibody can be humanized or chimeric. The antibody can be a naked antibody or a fusion antibody.

[0086] An antibody can be a full-length (i.e., naturally occurring or formed by conventional immunoglobulin gene fragment recombination processes) immunoglobulin molecule (e.g., an IgG antibody), or an immunologically active (i.e., specific binding) portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment can be, for example, a portion of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. In some embodiments, an antibody fragment can bind to the same antigen recognized by the full-length antibody. Antibody fragments can include isolated fragments consisting of the variable regions of an antibody, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains are connected by a peptide linker ("scFv protein"). Exemplary antibodies can include, but are not limited to, antibodies against cancer cells, antibodies against viruses, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

[0087] Universal Adapter Primer A barcode may include one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific stochastic barcode, may include a universal adapter primer. A universal adapter primer may refer to a nucleotide sequence that is universal across all barcodes. A universal adapter primer can be used to construct a gene-specific barcode. A universal adapter primer may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides in length, or a number or range between any two of these, or approximately these values ​​or such number or range of nucleotides. The universal adapter primer can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The universal adapter primer can be 5 to 30 nucleotides in length.

[0088] Linker When a barcode includes more than one type of label (e.g., more than one cellular label or more than one barcode sequence, e.g., one molecular label), the labels may be interspersed with linker label sequences. The linker label sequence may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides in length. The linker label sequence may be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides in length. In some cases, the linker label sequence is 12 nucleotides in length. The linker label sequence may be used to facilitate synthesis of the barcode. The linker label may include an error-correcting (e.g., Hamming) code.

[0089] solid support In some embodiments, the barcodes disclosed herein, e.g., stochastic barcodes, may be associated with a solid support. The solid support may be, for example, a synthetic particle. In some embodiments, some or all of the barcode sequences, e.g., molecular labels of stochastic barcodes (e.g., first barcode sequences) of a plurality of barcodes (e.g., a first plurality of barcodes) on a solid support, differ by at least one nucleotide. Cell labels of barcodes on the same solid support may be the same. Cell labels of barcodes on different solid supports may differ by at least one nucleotide. For example, a first cell label of a first plurality of barcodes on a first solid support may have the same sequence, and a second cell label of a second plurality of barcodes on a second solid support may have the same sequence. A first cell label of a first plurality of barcodes on a first solid support and a second cell label of a second plurality of barcodes on a second solid support may differ by at least one nucleotide. Cell labels may be, for example, about 5 to 20 nucleotides in length. The barcode sequence can be, for example, about 5-20 nucleotides in length. The synthetic particle can be, for example, a bead.

[0090] The beads can be, for example, silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The beads can include materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic materials, ceramic, plastic, glass, methylstyrene, acrylic polymers, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof. In some embodiments, the beads can be polymer beads, such as deformable beads or gel beads (e.g., gel beads from 10X Genomics (San Francisco, CA)) functionalized with barcodes or stochastic barcodes. In some implementations, the gel beads can comprise a polymer-based gel. Gel beads can be generated, for example, by encapsulating one or more polymer precursors into droplets. Gel beads can be generated when the polymer precursors are exposed to an accelerator (e.g., tetramethylethylenediamine (TEMED)).

[0091] The particles can be disintegrable (e.g., dissolvable, degradable). For example, polymer beads can dissolve, melt, or decompose under desired conditions. The desired conditions can include environmental conditions. The desired conditions can result in the dissolution, melt, or decomposition of the polymer beads in a controlled manner. Gel beads can dissolve, melt, or decompose due to a chemical stimulus, a physical stimulus, a biological stimulus, a thermal stimulus, a magnetic stimulus, an electrical stimulus, a light stimulus, or any combination thereof.

[0092] Analytes and / or reagents, e.g., oligonucleotide barcodes, may be linked / immobilized, for example, to the interior surface of a gel bead (e.g., the interior accessible through diffusion of the oligonucleotide barcodes and / or the material used to generate the oligonucleotide barcodes) and / or to the exterior surface of a gel bead or any other microcapsule described herein. Linkage / immobilization may be via any form of chemical bond (e.g., covalent bond, ionic bond) or physical phenomenon (e.g., van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, the linkage / immobilization of reagents to a gel bead or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical crosslinker, including those described herein). Upon application of a stimulus, the labile moiety can be cleaved, releasing the immobilized reagent. In some embodiments, the labile moiety is a disulfide bond. For example, in cases where an oligonucleotide barcode is immobilized to a gel bead via a disulfide bond, exposing the disulfide bond to a reducing agent can cleave the disulfide bond and release the oligonucleotide barcode from the bead. The labile moiety may be included as part of the gel bead or microcapsule, as part of a chemical linker connecting the reagent or analyte to the gel bead or microcapsule, and / or as part of the reagent or analyte. In some embodiments, at least one barcode of the plurality of barcodes may be immobilized to the particle, partially immobilized to the particle, encapsulated in the particle, partially encapsulated in the particle, or any combination thereof.

[0093] Gel beads can comprise a wide variety of different polymers, including but not limited to polymers, thermosensitive polymers, light-sensitive polymers, magnetic polymers, pH-sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. Polymers can include, but are not limited to, materials such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrenesulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(diallyldimethylammonium chloride) (PDADMAC), poly(pyrrole) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthalaldehyde) (PPA), poly(hexylviologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), and poly(lactic-co-glycolic acid) (PLGA).

[0094] A number of chemical stimuli can be used to trigger bead collapse, dissolution, or degradation. Examples of these chemical changes include, but are not limited to, pH-mediated changes to the bead wall, bead wall collapse via chemical cleavage of cross-links, triggering bead wall depolymerization, and bead wall switching reactions. Bulk changes can also be used to trigger bead collapse. Bulk or physical changes to microcapsules through various stimuli also offer many advantages in designing capsules for releasing reagents. Bulk or physical changes occur on a macroscopic scale, with bead rupture being the result of mechanical-physical forces induced by the stimulus. These processes can include, but are not limited to, pressure-induced rupture, bead wall melting, or changes in bead wall porosity.

[0095] Biological stimuli can also be used to trigger bead disintegration, dissolution, or degradation. Generally, biological triggers resemble chemical triggers, but many examples use biomolecules, or molecules commonly found in biological systems, such as enzymes, peptides, sugars, fatty acids, nucleic acids, and the like. For example, beads can contain polymers with peptide crosslinks that are susceptible to cleavage by specific proteases. More specifically, one example can include microcapsules containing GFLGK peptide crosslinks. Addition of a biological trigger, such as the protease cathepsin B, cleaves the peptide crosslinks in the shell wall, releasing the contents of the bead. In other cases, the protease can be heat-activated. In another example, beads contain a shell wall containing cellulose. Addition of the hydrolytic enzyme chitosan serves as a biological trigger for cleavage of the cellulose bonds, depolymerization of the shell wall, and release of its contents.

[0096] Beads can also be induced to release their contents upon application of a thermal stimulus. A change in temperature can cause various changes in the beads. A change in heat can cause the beads to melt, causing the bead walls to collapse. In other cases, heat can increase the internal pressure of the beads' internal components, causing the beads to collapse or explode. In still other cases, heat can transform the beads into a compressed, dehydrated state. Heat can also act on heat-sensitive polymers within the bead walls, causing the beads to collapse. The inclusion of magnetic nanoparticles in the bead walls of microcapsules can trigger the collapse of the beads as well as guide the beads in an array. The devices of the present disclosure can include magnetic beads for either purpose. In one example, the incorporation of Fe3O4 nanoparticles into polyelectrolyte-containing beads triggers collapse in the presence of an oscillating magnetic field stimulus.

[0097] Beads can also be disintegrated, dissolved, or decomposed as a result of electrical stimulation. Similar to the magnetic particles described in the previous section, electrically sensitive beads can trigger both bead disintegration and other functions, such as alignment in an electric field, electrical conduction, or redox reactions. In one example, beads containing electrically sensitive materials are aligned in an electric field to control the release of internal reagents. In another example, an electric field can induce redox reactions within the bead wall itself, which can increase porosity. Light stimulation can also be used to disrupt the beads. Numerous optical triggers are possible, including systems using various molecules such as nanoparticles and chromophores that can absorb photons of specific wavelengths. For example, metal oxide coatings can be used as capsule triggers. UV irradiation of SiO2-coated polyelectrolyte capsules can result in the collapse of the bead wall. In yet another example, photoswitchable materials, such as azobenzene groups, can be incorporated into the bead wall. Upon application of UV or visible light, chemicals such as these absorb photons and undergo reversible cis-to-trans isomerization. In this embodiment, the incorporation of a photoswitch results in a bead wall that can collapse or become more porous upon application of a light trigger. For example, in a non-limiting example of barcoding (e.g., stochastic barcoding) shown in FIG. 2, after cells, e.g., single cells, are introduced into multiple microwells of a microwell array in block 208, beads can be introduced into multiple microwells of the microwell array in block 212. Each microwell can contain one bead. The beads can contain multiple barcodes. The barcodes can include 5' amine regions attached to the beads. The barcodes can include a universal label, a barcode sequence (e.g., a molecular label), a target binding region, or any combination thereof.

[0098] The barcodes disclosed herein may be associated with (e.g., attached to) a solid support (e.g., a bead). The barcodes associated with the solid support may comprise a barcode sequence selected from a group comprising at least 100 or 1000 barcode sequences, each having a unique sequence. In some embodiments, different barcodes associated with a solid support may comprise barcodes with different sequences. In some embodiments, a percentage of the barcodes associated with a solid support comprise the same cell label. For example, the percentage may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or may be approximately these values ​​or such a number or range. As another example, the percentage may be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, barcodes associated with a solid support may have the same cell label. Barcodes associated with different solid supports may have different cell labels selected from a group comprising at least 100 or 1000 cell labels having unique sequences.

[0099] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., a bead). In some embodiments, barcoding a plurality of labels in a sample can be performed using a solid support comprising a plurality of synthetic particles associated with a plurality of barcodes. In some embodiments, the solid support may comprise a plurality of synthetic particles associated with a plurality of barcodes. The spatial labeling of the plurality of barcodes on different solid supports may differ by at least one nucleotide. The solid support may comprise a plurality of barcodes, for example, in two or three dimensions. The synthetic particles may be beads. The beads may be silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The solid support may include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the solid support may be free-floating. In some embodiments, the solid support may be embedded in a semi-solid or solid array. The barcodes may not be associated with a solid support. The barcodes may be individual nucleotides. The barcode may be associated with the substrate.

[0100] As used herein, the terms "tethered," "attached," and "immobilized" are used interchangeably and can refer to covalent or non-covalent means for attaching a barcode to a solid support. Any of a variety of different solid supports can be used to attach pre-synthesized barcodes or as a solid support for in situ solid phase synthesis of barcodes. In some embodiments, the solid support is a bead. Beads may include one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar structures to which nucleic acids can be immobilized (e.g., covalently or non-covalently). Beads may be composed of, for example, plastic, ceramic, metal, polymeric material, or any combination thereof. Beads may be or include discrete particles that are spherical (e.g., microspheres), or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. In some embodiments, beads may be non-spherical in shape.

[0101] The beads may comprise a variety of materials, including, but not limited to, paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (Fe3O4, magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, some alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, sepharose, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof. In some embodiments, the beads (e.g., the beads to which the label is attached) are hydrogel beads. In some embodiments, the beads comprise a hydrogel.

[0102] Some embodiments disclosed herein include one or more particles (e.g., beads). Each of the particles may include a plurality of oligonucleotides (e.g., barcodes). Each of the plurality of oligonucleotides may include a barcode sequence (e.g., a molecular label sequence), a cell label, and a target binding region (e.g., an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof). The cell label sequence of each of the plurality of oligonucleotides may be the same. The cell label sequences of oligonucleotides on different particles may be different so that the oligonucleotides on different particles can be identified. The number of different cell label sequences may vary in different implementations. In some embodiments, the number of cell labeling sequences is 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 In some embodiments, the number of cell labeling sequences may be at least or up to 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more of the plurality of particles comprise oligonucleotides having the same cellular sequence. In some embodiments, up to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more of the plurality of particles comprise oligonucleotides with the same cellular sequence, hi some embodiments, none of the plurality of particles have the same cellular targeting sequence.

[0103] The multiple oligonucleotides on each particle can include different barcode sequences (e.g., molecular labels). In some embodiments, the number of barcode sequences is 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9In some embodiments, the number of barcode sequences may be at least or at most 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 For example, at least 100 of the plurality of oligonucleotides comprise different barcode sequences. As another example, in a single particle, at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, a number or range between any two of these values, or more of the plurality of oligonucleotides comprise different barcode sequences. Some embodiments provide a plurality of particles comprising barcodes. In some embodiments, the ratio of occurrences (or copies or numbers) of targets to be labeled to distinct barcode sequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both. The particles can be, for example, nanoparticles or microparticles.

[0104] The size of the beads can vary. For example, the diameter of the beads can range from 0.1 micrometers to 50 micrometers. In some embodiments, the diameter of the beads can be 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or range between any two of these values, or can be approximately these values ​​or such numbers or ranges. The diameter of the beads may be related to the diameter of the wells of the substrate. In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values, longer or shorter than the diameter of the wells, or may be approximately these values ​​or any such number or range, longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of a cell (e.g., a single cell surrounded by a well of the substrate). In some embodiments, the diameter of the beads may be at least or up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of a cell (e.g., a single cell surrounded by a well of the substrate). In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or range between any two of these values, longer or shorter than the diameter of the cells, or may be approximately these values ​​or any such number or range, longer or shorter than the diameter of the cells. In some embodiments, the diameter of the beads may be at least or up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% longer or shorter than the diameter of the cells.

[0105] The beads may be bound and / or embedded in a substrate. The beads may be bound and / or embedded in a gel, hydrogel, polymer, and / or matrix. The spatial location of the beads within the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using spatial labels present in barcodes on the beads, which can serve as locational addresses. Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® microbeads, antibody-conjugated beads (e.g., anti-immunoglobulin microbeads), protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, and BcMag™ carboxyl-terminated magnetic beads.

[0106] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make them fluorescent in one fluorescent optical channel or multiple optical channels. The beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The beads can be identifiable. For example, the beads can be imaged using a camera. The beads can have a detectable code associated with them. For example, the beads can include a barcode. The beads can change size, for example, due to swelling in an organic or inorganic solution. The beads can be hydrophobic. The beads can be hydrophilic. The beads can be biocompatible. The solid support (e.g., beads) can be visualized. The solid support can include a visualization tag (e.g., a fluorescent dye). The solid support (e.g., beads) can be etched with an identifier (e.g., a number). The identifier can be visualized through imaging of the beads.

[0107] A solid support can comprise an insoluble, semi-soluble, or insoluble material. A solid support can be referred to as "functionalized" if it includes a linker, scaffold, building block, or other reactive moiety attached thereto, while a solid support can be "non-functionalized" if it lacks such reactive moieties attached thereto. A solid support can be freely used in solution, e.g., in a microtiter well format, in a flow-through format, e.g., in a column, or in a dipstick.

[0108] The solid support may comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof. The solid support may take the form of a resin, gel, microsphere, or other geometric configuration. The solid support may comprise a silica chip, microparticle, nanoparticle, plate, array, caliper, flat support, such as a glass fiber filter, glass surface, metal surface, metal surface (steel, gold silver, aluminum, silicone, and copper), glass support, plastic support, silicone support, chip, filter, membrane, microwell plate, slide, multiwell plate, or plastic material (e.g., formed from polyethylene, polypropylene, polyamide, polyvinylidene difluoride), including membrane, and / or wafer, comb, pin, or needle (e.g., an array of pins suitable for combinatorial synthesis or analysis), or an array of holes or nanoliter wells on a flat surface, such as a wafer (e.g., a silicone wafer), a wafer with holes or without a filter bottom, or beads. The solid support can include a polymer matrix (e.g., a gel, a hydrogel). The polymer matrix can be capable of penetrating intracellular spaces (e.g., around organelles). The polymer matrix can be capable of being pumped through the circulatory system.

[0109] Substrates and Microwell Arrays As used herein, a substrate may refer to a type of solid support. A substrate may refer to a solid support that may include a barcode or stochastic barcode of the present disclosure. A substrate may include, for example, a plurality of microwells. For example, a substrate may be a well array including two or more microwells. In some embodiments, a microwell may include a small reaction chamber having a defined volume. In some embodiments, a microwell may incorporate one or more cells. In some embodiments, a microwell may incorporate only one cell. In some embodiments, a microwell may incorporate one or more solid supports. In some embodiments, a microwell may incorporate only one solid support. In some embodiments, a microwell incorporates a single cell and a single solid support (e.g., a bead). A microwell may include a barcode reagent of the present disclosure.

[0110] Barcoding methods The present disclosure provides a method for estimating the number of distinct targets in distinct locations of a body sample (e.g., tissue, organ, tumor, cell). The method may include placing a barcode (e.g., a stochastic barcode) in proximity to the sample, lysing the sample, associating distinct targets with the barcode, amplifying the targets, and / or digitally counting the targets. The method may further include analyzing and / or visualizing information obtained from the spatial labeling of the barcode. The method may include visualizing multiple targets in the sample. Mapping the multiple targets to a map of the sample may include creating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps may be created before or after barcoding (e.g., stochastically barcoding) the multiple targets in the sample. Visualizing multiple targets in the sample may include mapping the multiple targets to a map of the sample. Mapping the multiple targets to a map of the sample may include creating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps can be generated before or after barcoding multiple targets in a sample. In some embodiments, the two-dimensional and three-dimensional maps can be generated before or after lysing the sample. Lysing the sample before or after generating the two-dimensional or three-dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0111] In some embodiments, barcoding the plurality of targets comprises hybridizing a plurality of barcodes to the plurality of targets to create barcoded targets (e.g., stochastically barcoded targets). Barcoding the plurality of targets may comprise generating an indexed library of barcoded targets. Generating an indexed library of barcoded targets may be performed using a solid support comprising a plurality of barcodes (e.g., stochastic barcodes).

[0112] Contacting the sample with the barcode The present disclosure provides methods for contacting a sample (e.g., cells) with a substrate of the present disclosure. For example, a sample including cells, an organ, or a thin section of tissue can be contacted with a barcode (e.g., a stochastic barcode). For example, the cells can be contacted by gravity flow, where they can settle and form a monolayer. The sample can be a tissue slice. The slice can be disposed on a substrate. The sample can be one-dimensional (e.g., forming a planar surface). For example, the sample (e.g., cells) can be spread across the substrate by growing / culturing the cells on the substrate. When the barcode is in close proximity to the target, the target can hybridize to the barcode. The barcodes can be contacted in a non-depleting ratio so that each distinct target can associate with a distinct barcode of the present disclosure. To ensure efficient association between the target and the barcode, the target can be cross-linked to the barcode.

[0113] Cell lysis After partitioning the cells and barcodes, the cells can be lysed to liberate the target molecules. Cell lysis can be achieved by any of a variety of means, such as chemical or biochemical means, osmotic shock, or thermal, mechanical, or optical lysis. Cells can also be lysed by adding a cell lysis buffer containing a detergent (e.g., SDS, Li-dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or a digestive enzyme (e.g., proteinase K, pepsin, or trypsin), or any combination thereof. To increase the association of the target with the barcode, the diffusion rate of the target molecule can be altered, for example, by lowering the temperature and / or increasing the viscosity of the lysate.

[0114] In some embodiments, the sample may be lysed using filter paper, which can be soaked with a lysis buffer over the filter paper, and pressure can be applied to the sample, which can promote lysis of the sample and hybridization of the sample's targets to the substrate. In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis can include the use of digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer can include Tris-HCl. The lysis buffer can include at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris-HCl. The lysis buffer can include up to about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris-HCl. The lysis buffer can include about 0.1 M Tris-HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer may include a salt (e.g., LiCl). The salt concentration in the lysis buffer may be at least about 0.1, 0.5, or 1 M or higher. The salt concentration in the lysis buffer may be up to about 0.1, 0.5, or 1 M or higher. In some embodiments, the salt concentration in the lysis buffer is about 0.5 M. The lysis buffer may include a detergent (e.g., SDS, Li-dodecyl sulfate, triton X, tween, NP-40). The concentration of the detergent in the lysis buffer may be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. The concentration of detergent in the lysis buffer may be up to about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7%, or higher. In some embodiments, the concentration of detergent in the lysis buffer is about 1% Li-dodecyl sulfate. The time used in the lysis method may depend on the amount of detergent used. In some embodiments, the more detergent used, the less time is required for lysis.The lysis buffer may include a chelating agent (e.g., EDTA and EGTA). The concentration of the chelating agent in the lysis buffer may be at least about 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of the chelating agent in the lysis buffer may be up to about 1, 5, 10, 15, 20, 25, or 30 mM or more. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10 mM. The lysis buffer may include a reducing reagent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing reagent in the lysis buffer may be at least about 1, 5, 10, 15, or 20 mM or more. The concentration of the reducing reagent in the lysis buffer may be up to about 1, 5, 10, 15, or 20 mM or more. In some embodiments, the concentration of the reducing reagent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer may comprise about 0.1 M Tris-HCl (about pH 7.5), about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

[0115] Lysing can be performed at a temperature of about 4, 10, 15, 20, 25, or 30° C. Lysing can be performed for about 1, 5, 10, 15, 20 minutes, or longer. Lysed cells may contain at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules. Lysed cells may contain up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules.

[0116] Attaching the barcode to the target nucleic acid molecule After cell lysis and release of nucleic acid molecules therefrom, the nucleic acid molecules may randomly associate with the barcodes on the co-localized solid support. Association may involve hybridization of the target recognition region of the barcode to a complementary portion of the target nucleic acid molecule (e.g., the oligo(dT) of the barcode may interact with the poly(A) tail of the target). Assay conditions (e.g., buffer pH, ionic strength, temperature, etc.) used for hybridization may be selected to promote the formation of specific, stable hybrids. In some embodiments, nucleic acid molecules released from lysed cells may associate with multiple probes on a substrate (e.g., hybridize to the probes on a substrate). If the probes contain oligo(dT), mRNA molecules may hybridize to the probes and be reverse transcribed. The oligo(dT) portion of the oligonucleotide may act as a primer for first-strand synthesis of cDNA molecules. For example, in the non-limiting example of barcoding shown in block 216 of FIG. 2, mRNA molecules may hybridize to barcodes on beads. For example, a single-stranded nucleotide fragment can hybridize to the target binding region of the barcode.

[0117] The binding may further include ligating the target recognition region of the barcode with a portion of the target nucleic acid molecule. For example, the target binding region may include a nucleic acid sequence capable of specific hybridization to a restriction site overhang (e.g., an EcoRI sticky end overhang). The assay procedure may further include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to generate a restriction site overhang. The barcode can then be ligated to any nucleic acid molecule that contains a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to connect the two fragments.

[0118] For example, in a non-limiting example of barcoding shown in block 220 of Figure 2, labeled targets (e.g., target-barcode molecules) from multiple cells (or multiple samples) can then be pooled, e.g., in a tube. For example, the labeled targets can be pooled by collecting beads to which barcodes and / or target-barcode molecules are bound. Solid support-based collection recovery of bound target-barcode molecules can be achieved through the use of magnetic beads and an externally applied magnetic field. Once the target-barcode molecules are pooled, all further processing can proceed within a single reaction vessel. Further processing can include, for example, reverse transcription, amplification, cleavage, dissociation, and / or nucleic acid extension reactions. Further processing reactions can be performed within microwells, i.e., without first pooling labeled target nucleic acid molecules from multiple cells.

[0119] Reverse transcription or nucleic acid extension The present disclosure provides methods for generating target-barcode conjugates using reverse transcription (e.g., block 224 of Figure 2) or nucleic acid extension. The target-barcode conjugates can include a barcode and a complementary sequence of all or a portion of a target nucleic acid (i.e., a barcoded cDNA molecule, e.g., a stochastically barcoded cDNA molecule). Reverse transcription of the associated RNA molecule can occur by adding a reverse transcription primer along with a reverse transcriptase. The reverse transcription primer can be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The oligo(dT) primer can be 12-18 nucleotides in length, or about 12-18 nucleotides in length, and binds to the endogenous poly(A) tail at the 3' end of mammalian mRNA. The random hexanucleotide primer can bind to the mRNA at various complementary sites. The target-specific oligonucleotide primer typically selectively primes the mRNA of interest.

[0120] In some embodiments, reverse transcription of mRNA molecules into labeled RNA molecules can occur by the addition of a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. Typically, oligo(dT) primers are 12-18 nucleotides in length and bind to the endogenous poly(A) tail at the 3' end of mammalian mRNAs. Random hexanucleotide primers can bind to mRNAs at various complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest. The target can be, for example, a cDNA molecule. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase such as Moloney murine leukemia virus (MMLV) reverse transcriptase to generate a cDNA molecule with a poly(dC) tail. The barcode can include a target binding region with a poly(dG) tail. Upon base pairing between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues replication to the 5' end of the barcode. The resulting cDNA molecule then contains the barcode sequence (e.g., a molecular tag) at the 3' end of the cDNA molecule. Reverse transcription can occur repeatedly to generate multiple labeled cDNA molecules. The methods disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The methods can include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0121] amplification One or more nucleic acid amplification reactions (e.g., block 228 of FIG. 2 ) can be performed to generate multiple copies of the labeled target nucleic acid molecule. Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. The amplification reaction can be used to add sequencing adapters to the nucleic acid molecule. The amplification reaction can include amplifying at least a portion of the sample label, if present. The amplification reaction can include amplifying at least a portion of the cell label and / or barcode sequence (e.g., molecular label). The amplification reaction can include amplifying at least a portion of the sample tag, cell label, spatial label, barcode sequence (e.g., molecular label), target nucleic acid, or a combination thereof. The amplification reaction may include amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100%, or a range or number between any two of these values ​​of the plurality of nucleic acids. The method may further include performing one or more cDNA synthesis reactions to generate one or more cDNA copies of the target-barcode molecule comprising the sample label, cell label, spatial label, and / or barcode sequence (e.g., molecular label).

[0122] In some embodiments, amplification can be performed using polymerase chain reaction (PCR). As used herein, PCR can refer to a reaction for amplifying specific DNA sequences in vitro by simultaneous primer extension of complementary strands of DNA. As used herein, PCR can encompass derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, and assembly PCR.

[0123] Amplification of labeled nucleic acids may include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiplex displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-circle amplification. Other non-PCR-based amplification methods include DNA-dependent RNA polymerase-driven RNA transcription amplification or multiple cycles of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), and Qβ replicase (Qβ) methods, the use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved before extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and branched extension amplification (RAM). In some embodiments, amplification does not produce circularized transcripts.

[0124] In some embodiments, the methods disclosed herein further include performing a polymerase chain reaction on the labeled nucleic acid (e.g., labeled RNA, labeled DNA, labeled cDNA) to generate labeled amplicons (e.g., stochastically labeled amplicons). The labeled amplicons may be double-stranded molecules. The double-stranded molecules may comprise double-stranded RNA molecules, double-stranded DNA molecules, or RNA molecules hybridized to DNA molecules. One or both strands of the double-stranded molecules may comprise a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicons may be single-stranded molecules. The single-stranded molecules may comprise DNA, RNA, or a combination thereof. The nucleic acids of the present disclosure may include synthetic or modified nucleic acids.

[0125] Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile or trigger nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides may be used to identify products at specific cycles or time points of the amplification reaction.

[0126] Performing one or more amplification reactions may include the use of one or more primers. The one or more primers may contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may contain fewer than 12 to 15 nucleotides. The one or more primers may anneal to at least a portion of the multiple labeled targets (e.g., stochastically labeled targets). The one or more primers may anneal to the 3' or 5' ends of the multiple labeled targets. The one or more primers may anneal to an internal region of the multiple labeled targets. The internal region may be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the multiple labeled targets. The one or more primers may comprise a constant panel of primers. The one or more primers may include at least one or more custom primers, the one or more primers may include at least one or more control primers, or the one or more primers may include at least one or more gene-specific primers.

[0127] The one or more primers may include a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to a first sample label, a second sample label, a spatial label, a cell label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof. The one or more primers may include a universal primer and a custom primer. The custom primers may be designed to amplify one or more targets. The targets may comprise a subset of all nucleic acids in one or more samples. The targets may comprise a subset of all labeled targets in one or more samples. The one or more primers may include at least 96 or more custom primers. The one or more primers may include at least 960 or more custom primers. The one or more primers may include at least 9600 or more custom primers. The one or more custom primers may anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids may correspond to one or more genes.

[0128] Any amplification scheme can be used in the disclosed methods. For example, in one scheme, a first PCR can amplify the molecules bound to the beads using a gene-specific primer and a primer for the sequence of universal Illumina sequencing primer 1. A second PCR can amplify the first PCR product using a nested gene-specific primer adjacent to the sequence of Illumina sequencing primer 2 and a primer for the sequence of universal Illumina sequencing primer 1. A third PCR adds P5 and P7 and a sample index to place the PCR product into an Illumina sequencing library. Sequencing using 150 bp x 2 sequencing can reveal cell label and barcode sequences (e.g., molecular labels) on read 1, genes on read 2, and a sample index on index 1 read.

[0129] In some embodiments, chemical cleavage can be used to remove nucleic acids from a substrate. For example, chemical groups or modified bases present in the nucleic acid can be used to facilitate its removal from a solid support. For example, enzymes can be used to remove nucleic acids from a substrate. For example, nucleic acids can be removed from a substrate by restriction endonuclease digestion. For example, nucleic acids containing dUTP or ddUTP can be removed from a substrate using uracil-d-glycosylase (UDG) treatment. For example, enzymes that perform nucleotide excision, such as base excision repair enzymes, for example, apurinic / apyrimidinic (AP) endonucleases, can be used to remove nucleic acids from a substrate. In some embodiments, photocleavable groups and light can be used to remove nucleic acids from a substrate. In some embodiments, a cleavable linker can be used to remove nucleic acids from a substrate. For example, the cleavable linker can comprise at least one of biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig-Protein A, a photolabile linker, an acid or base labile linker group, or an aptamer.

[0130] If the probe is gene-specific, the molecule can be hybridized to the probe and reverse transcribed and / or amplified. In some embodiments, the nucleic acid can be amplified after it is synthesized (e.g., reverse transcribed). Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adapters to the nucleic acid.

[0131] In some embodiments, amplification can be performed on the substrate using, for example, bridge amplification. Homopolymer tails can be added to cDNA to generate ends compatible with bridge amplification using oligo(dT) probes on the substrate. In bridge amplification, a primer complementary to the 3' end of the template nucleic acid can be the first primer of each pair covalently attached to solid particles. When a sample containing the template nucleic acid is contacted with the particles and a single thermal cycle is performed, the template molecule anneals to the first primer, and the first primer can be extended in the forward direction by adding nucleotides to form a double-stranded molecule consisting of the template molecule and a newly formed DNA strand complementary to the template. In the heating step of the next cycle, the double-stranded molecule can be denatured, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle through the first primer. In the annealing stage of the subsequent annealing and extension step, the complementary strand can hybridize to a second primer complementary to the segment of the complementary strand at the position removed from the first primer. Through this hybridization, the complementary strand can form a bridge between the first and second primers, immobilized by covalent bonding to the first primer and by hybridization to the second primer. In the extension step, the second primer can be extended in the opposite direction by adding nucleotides to the same reaction mixture, thereby converting the bridge into a double-stranded bridge. The next cycle then begins, and the double-stranded bridge is denatured to yield two single-stranded nucleic acid molecules, each with one end bound to the particle surface via the first and second primers, and the other end unbound. In the annealing and extension step of this second cycle, each strand can hybridize to a previously unused additional complementary primer on the same particle to form a new single-stranded bridge. The two previously unused hybridized primers then extend, converting the two new bridges into double-stranded bridges.

[0132] The amplification reaction can include amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100% of the plurality of nucleic acids. Amplification of the labeled nucleic acid may include PCR-based or non-PCR-based methods. Amplification of the labeled nucleic acid may include exponential amplification of the labeled nucleic acid. Amplification of the labeled nucleic acid may include linear amplification of the labeled nucleic acid. Amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for in vitro amplification of specific DNA sequences by simultaneous primer extension of complementary strands of DNA. PCR may encompass derivative forms of the reaction, including, but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, suppression PCR, semi-suppressive PCR, and assembly PCR.

[0133] In some embodiments, amplification of the labeled nucleic acid includes non-PCR-based methods such as MDA, TMA, NASBA, SDA, real-time SDA, rolling circle amplification, circle-circle amplification, multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ), use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and / or branched extension amplification (RAM).

[0134] In some embodiments, the methods disclosed herein further comprise performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon may be a double-stranded molecule. The double-stranded molecule may comprise a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule may comprise a sample tag or molecular identifier label. Alternatively, the amplicon may be a single-stranded molecule. The single-stranded molecule may comprise DNA, RNA, or a combination thereof. The nucleic acids of the invention may comprise synthetic or modified nucleic acids. In some embodiments, the methods include repeatedly amplifying a labeled nucleic acid to generate multiple amplicons. The methods disclosed herein may include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the methods include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0135] The amplification may further include adding one or more control nucleic acids to one or more samples containing the plurality of nucleic acids. The amplification may further include adding one or more control nucleic acids to the plurality of nucleic acids. The control nucleic acids may include a control label. Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile and / or trigger nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides may be used to identify products at specific cycles or time points of the amplification reaction.

[0136] Performing one or more amplification reactions may involve the use of one or more primers. The one or more primers may comprise one or more oligonucleotides. The one or more oligonucleotides may comprise at least about 7 to 9 nucleotides. The one or more oligonucleotides may comprise fewer than 12 to 15 nucleotides. The one or more primers may anneal to at least a portion of the plurality of labeled nucleic acids. The one or more primers may anneal to the 3' and / or 5' ends of the plurality of labeled nucleic acids. The one or more primers may anneal to an internal region of the plurality of labeled nucleic acids. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides from the 3' end of the plurality of labeled nucleic acids. The one or more primers can comprise a fixed panel of primers. The one or more primers may include at least one or more custom primers. The one or more primers may include at least one or more control primers. The one or more primers may include at least one or more housekeeping gene primers. The one or more primers may include a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to a first sample tag, a second sample tag, a molecular identifier label, a nucleic acid, or a product thereof. The one or more primers may include a universal primer and a custom primer. The custom primer may be designed to amplify one or more target nucleic acids. The target nucleic acids may comprise a subset of the total nucleic acids in one or more samples. In some embodiments, the primers are probes bound to an array of the present disclosure.

[0137] In some embodiments, barcoding (e.g., stochastically barcoding) a plurality of targets in a sample further includes generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) or barcoded fragments of those targets. The barcode sequences of different barcodes (e.g., molecular labels of different stochastic barcodes) may differ from each other. Generating an indexed library of barcoded targets includes generating a plurality of indexed polynucleotides from the plurality of targets in the sample. For example, for an indexed library of barcoded targets including a first indexed target and a second indexed target, the labeled region of the first indexed polynucleotide may differ from the labeled region of the second indexed polynucleotide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or a number or range between, or about, or at least, or at most, any two of these values. In some embodiments, generating an indexed library of barcoded targets includes contacting a plurality of targets, e.g., mRNA molecules, with a plurality of oligonucleotides comprising a poly(T) region and a label region, and performing first-strand synthesis using a reverse transcriptase to generate single-stranded, labeled cDNA molecules, each comprising a cDNA region and a label region, wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences. Generating an indexed library of barcoded targets may further include amplifying the single-stranded, labeled cDNA molecules to generate double-stranded, labeled cDNA molecules, and performing nested PCR on the double-stranded, labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method may include generating adapter-labeled amplicons.

[0138] Barcoding (e.g., stochastic barcoding) can include labeling individual nucleic acid (e.g., DNA or RNA) molecules with nucleic acid barcodes or tags. In some embodiments, this includes adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can minimize PCR amplification bias. Adapters can be added for sequencing, e.g., using next-generation sequencing (NGS). For example, sequencing results can be used to determine the sequence of cellular labels, molecular labels, and nucleotide fragments of one or more copies of the target in block 232 of FIG. 2.

[0139] 3 is a schematic diagram illustrating a non-limiting, exemplary process for generating an indexed library of barcoded targets (e.g., stochastically barcoded targets), e.g., barcoded mRNAs or fragments thereof. As shown in step 1, a reverse transcription process can encode each mRNA molecule containing a unique molecular label sequence, a cellular label sequence, and a universal PCR site. In particular, the RNA molecule 302 can be reverse transcribed to generate labeled cDNA molecules 304 containing cDNA regions 306 by hybridization (e.g., stochastic hybridization) of a set of barcodes (e.g., stochastic barcodes) 310 to a poly(A) tail region 308 of the RNA molecule 302. Each of the barcodes 310 can include a target binding region, e.g., a poly(dT) region 312, a label region 314 (e.g., a barcode sequence or molecule), and a universal PCR region 316.

[0140] In some embodiments, the cell label sequence may comprise 3 to 20 nucleotides. In some embodiments, the molecular label sequence may comprise 3 to 20 nucleotides. In some embodiments, each of the plurality of stochastic barcodes further comprises one or more of a universal label and a cell label, wherein the universal label is the same for the plurality of stochastic barcodes on the solid support and the cell label is the same for the plurality of stochastic barcodes on the solid support. In some embodiments, the universal label may comprise 3 to 20 nucleotides. In some embodiments, the cell label comprises 3 to 20 nucleotides.

[0141] In some embodiments, label region 314 may include a barcode sequence or molecular label 318 and a cell label 320. In some embodiments, label region 314 may include one or more of a universal label, a dimensional label, and a cell label. Barcode sequence or molecular label 318 may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between any of these values, in length, or may be approximately, at least, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in length between any of these values. A cell label 320 may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between, or may be approximately, or may be at least, or may be up to, these values ​​or such number or range of nucleotides in length. A universal label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between, or may be approximately, or may be at least, or may be up to, these values ​​or such number or range of nucleotides in length. The universal label may be the same for multiple stochastic barcodes on a solid support, and the cell label may be the same for multiple stochastic barcodes on a solid support.A dimension marker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides in between any of these values, in length, or may be approximately, at least, or at most, these values ​​or numbers or ranges of nucleotides in length.

[0142] In some embodiments, label region 314 may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any of these values, of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include approximately these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include at least these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include up to these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320. Each label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range between any of these values, in length, or may be approximately, at least, or at most these values ​​or number or range of nucleotides in length. A set of barcodes or stochastic barcodes 310 may be 10, 20, 40, 50, 70, 80, 90, 100, or a number or range between any of these values. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 107 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 , or a number or range of barcodes or stochastic barcodes 310 between any of these values, or about these values ​​or such number or range of barcodes or stochastic barcodes 310, or at least these values ​​or such number or range of barcodes or stochastic barcodes 310, or at most these values ​​or such number or range of barcodes or stochastic barcodes 310. The set of barcodes or stochastic barcodes 310 may also each contain, for example, a unique labeled region 314. The labeled cDNA molecules 304 may be purified to remove excess barcodes or stochastic barcodes 310. Purification may include Ampure bead purification.

[0143] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled in one tube and PCR amplified using a first pool of PCR primers and a first universal PCR primer. Pooling is possible due to the unique label region 314. In particular, the labeled cDNA molecules 304 can be amplified to generate nested PCR-labeled amplicons 322. The amplification can include multiplex PCR amplification. The amplification can include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, the multiplex PCR amplification can be performed using 10, 20, 40, 50, 70, 80, 90, 10, 25, 30, 45, 50, 60, 75, 80, 90, 100, 150, 250, 300, 450, 500, 600, 750, 800, 900, 1500, 1500, 2500, 3000, 4500, 5000, 6000, 15000, 25000, 30000, 45000, 50000, 50000, 60000, 75000, 8000, 9000, 15000, 15000, 15000, 25000, 30000, 45000, 50000, 15000, 25000, 30000, 45000, 50000, 60000, 150000, 250000, 300000, 450000, 50000, 60000, 150000, 25000 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 1013 , 10 14 , 10 15 , 10 20 , or a number or range of multiplex primers between any of these values, or approximately, or at least, or up to these values. The amplification may include a first PCR primer pool 324 including custom primers 326A-C targeting specific genes and a universal primer 328. The custom primer 326 may hybridize to a region within the cDNA portion 306' of the labeled cDNA molecule 304. The universal primer 328 may hybridize to the universal PCR region 316 of the labeled cDNA molecule 304.

[0144] As shown in step 3 of Figure 3, the product from the PCR amplification in step 2 can be amplified using a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, nested PCR-labeled amplicons 322 can be further amplified by nested PCR. Nested PCR can include multiplex PCR including a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328' in a single reaction volume. The nested PCR primer pool 328 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any of these values, of different nested PCR primers 330, or may contain about, or at least, or at most, of different nested PCR primers 330. The nested PCR primers 332 may contain an adaptor 334 and hybridize to a region within the cDNA portion 306" of the labeled amplicon 322. Universal primer 328' contains adapter 336 and can hybridize to universal PCR region 316 of labeled amplicon 322. Thus, step 3 generates adapter-labeled amplicon 338. In some embodiments, nested PCR primer 332 and second universal PCR primer 328' may not contain adapters 334 and 336. Instead, adapters 334 and 336 can ligate to the product of the nested PCR to generate adapter-labeled amplicon 338.

[0145] As shown in step 4, the PCR products from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, adapters 334 and 336 can be used to perform one or more additional assays on adapter-labeled amplicons 338. Adapters 334 and 336 can be hybridized with primers 340 and 342. One or more of primers 340 and 342 can be PCR amplification primers. One or more of primers 340 and 342 can be sequencing primers. One or more of adapters 334 and 336 can be used for further amplification of adapter-labeled amplicons 338. One or more of adapters 334 and 336 can be used for sequencing of adapter-labeled amplicons 338. Primer 342 can contain a plate index 344, which allows amplicons generated using the same set of barcodes or stochastic barcodes 310 to be sequenced in a single sequencing reaction using next-generation sequencing (NGS).

[0146] Methods and compositions for single cell secretomics The present disclosure includes methods for measuring the copy number of secreted analytes secreted by single cells. In some embodiments, compositions and methods are provided for bead-based single-cell secretome analysis in combination with CITE-seq / AbSeq using scRNA-seq (e.g., Rhapsody) systems. Single-cell secretome analysis methods and compositions using scRNA-seq (e.g., Rhapsody) components and / or workflows are used.

[0147] The disclosure herein includes single-cell secretome (scS) beads and methods of use. The compositions and methods provided herein enable characterization of proteins secreted by single cells (e.g., using the Rhapsody platform) and can be combined with single-cell multi-omics information (e.g., surface antigen and transcriptome data obtained by AbSeq / CITE-seq). The disclosed methods are compatible with current single-cell RNA-seq workflows (e.g., Rhapsody workflows using Rhapsody cartridges) with few additional steps. The compositions and methods provided herein complement current omics platforms. Embodiments of using AbS to determine protein expression profiles in single cells and track sample origin are described in U.S. Patent Application Publication Nos. 2018 / 0088112 and 2018 / 0346970, the contents of each of which are incorporated herein by reference in their entireties.

[0148] In some embodiments, scS beads (e.g., first solid supports) are provided, which may be approximately 0.5 μm in size and made of a ferromagnetic material. An antigen-specific capture antibody (e.g., a capture reagent) can be conjugated to the scS beads (e.g., first solid support). A single cell can be incubated with a large number of scS beads (e.g., first solid support) in a microwell of an scRNA-seq cartridge (e.g., a Rhapsody cartridge). Proteins secreted by the single cell can be captured by antibodies on the scS beads. Antigens captured on the scS beads (e.g., first solid support) can be detected by detection antibodies (e.g., secreted analyte-binding reagents) conjugated to specific oligonucleotides (e.g., secreted analyte-binding reagent-specific oligonucleotides). In some embodiments, subsequent steps can be the same as in the AbSeq / CITE-seq workflow. The methods and compositions provided herein can be highly sensitive and can quantify a large number of secreted proteins at the single-cell level.

[0149] In some embodiments, a single cell is incubated with a large number of scS beads (e.g., a first solid support). A capture antibody (e.g., a capture reagent) can be conjugated onto the scS beads (e.g., a first solid support). The scS beads can target various secreted products. The captured antigen can be detected by a detection antibody (e.g., a secreted analyte-binding reagent) conjugated with an oligo (e.g., a secreted analyte-binding reagent-specific oligonucleotide) and evaluated by current scRNA-seq (e.g., Rhapsody) workflows. Each detection antibody (e.g., a secreted analyte-binding reagent) can have a unique index sequence (e.g., a unique analyte identifier sequence for the secreted analyte-binding reagent) along with a primer-binding site (e.g., a universal sequence) and a polyA sequence. The poly-T sequence of the cell capture bead oligo (e.g., oligonucleotide barcode) can base pair with the poly-A of the detection antibody oligo, resulting in a product (e.g., a barcoded secreted analyte-binding reagent-specific oligonucleotide) that can have a unique sequence that identifies the cell and, if present, the secreted factor. The unique sequence from the cell capture bead (e.g., cell label) can be shared between the mRNA target cDNA, Ab-seq oligo, detection antibody Ab-seq oligo, and sample tag oligo from the same well.

[0150] In some embodiments, the single-cell secretome analysis workflow provided herein includes one or more of the following steps: (Step 1) scS beads (e.g., a first solid support) can be added to an scRNA-seq (e.g., Rhapsody) cartridge. The number of scS beads can be calculated using a Poisson distribution to obtain an average of 8 beads per well, thereby reducing wells without scS beads to 0.0003% (Table 1). In some embodiments, a mixture of scS beads is added to detect multiple secreted proteins, or different scS beads are added multiple times. (Step 2) Cells obtained from in vitro or ex-vivo culture can be contacted (e.g., mixed) with a second solid support (e.g., BD iMag beads) containing multiple isolation reagents targeted to the cells of interest. (Step 3) The single cells associated with the second solid support (e.g., cells with iMag beads bound) can be dispensed into an scRNA-seq (e.g., Rhapsody) cartridge to obtain single cells in a well (e.g., the same as in the current scRNA-seq (e.g., Rhapsody) workflow). (Step 4) The cells with scS beads (e.g., the first solid support) can be incubated in the scRNA-seq cartridge for 60–90 minutes. (Step 5) A detection antibody conjugated to a specific nucleotide tag (e.g., a secreted analyte-binding reagent conjugated to a secreted analyte-binding reagent-specific oligonucleotide) can be added and incubated for 20–30 minutes. (Step 6) The user can flush out unbound detection antibody by irrigating with 30–50 ml of wash buffer with a magnet in place. (Step 7) The user can dispense Rhapsody beads (e.g., the third solid support) and continue with the current scRNA-seq (e.g., Rhapsody) workflow, lysing the cells, and performing subsequent steps.

[0151] [Table 1]

[0152] Current methods for combining proteomics and transcriptomics are limited to surface antigens (e.g., CITE-seq). Intracellular staining of cytokines and other secreted factors involves blocking secretion and performing intracellular staining, which can interfere with the integrity of RNA transcripts. The disclosed compositions and methods leverage existing scRNA-seq (e.g., Rhapsody) systems, allowing users to simultaneously capture information on mRNA, surface proteins, and secreted substances.

[0153] Currently available methods use bispecific antibodies directed against CD45 and specific anticytokine antibodies. However, this method suffers from high background and crosstalk between cytokine-secreting cells and bystander cells. Furthermore, the number of secreted proteins that can be detected by this method is limited to a small number of antigens. While background noise is better when cells are rare, it still exists. This is significant for identifying antigen-specific cells and associated TCR- or antibody-producing B cells in downstream applications. Therefore, there is a need for alternative methods of single-cell secretome analysis. Currently available methods suffer from concerns about crosstalk between cells and secreted cytokines present in the supernatant. Furthermore, while surrogate surface markers are available for some intracellular or secreted factors, correlation is poor for the majority.

[0154] Currently available optical fluidic assays involve screening single cells and identifying cells of interest based on what they secrete (B cells), but are very time-consuming and labor-intensive and cannot be used to characterize large numbers of cells or to identify rare cells. Similar methods for detecting large numbers of secreted proteins are not available, especially for T cells. The disclosed compositions and methods allow for the parallel analysis of thousands of single cells—their secreted proteins and associated RNA transcripts. Currently available Isoplexis assays for identifying secreted proteins in single cells are low-throughput, and combining with transcriptome information involves additional steps. The disclosed compositions and methods enable the analysis of secretome, RNA transcript, and cellular protein information in a single workflow, and are ultrasensitive and quantitative.

[0155] The disclosed compositions and methods can be used to characterize CAR-T cells based on what they secrete, antigen markers, TCR sequences, and transcriptome profiles, all of which are important for assessing response and immunotherapy outcomes. The disclosed compositions and methods are also important for other applications, such as characterizing regulatory T cells, tumor-associated macrophages, and NK cells. Additional applications of the disclosed compositions and methods include characterizing cells differentiated from stem cell populations (e.g., cancer, regenerative medicine (based on what the cells secrete)). Secretome characterization (e.g., measuring cellular component targets) in conjunction with CITE-seq / AbSeq can expand biomarker development applications. In some embodiments, a first solid support (e.g., scS beads) can be directly conjugated to an antigen or antigen epitope / competitive assay and used to characterize B cells and / or identify antibody (e.g., neutralizing) sequences. Including single cells in an scRNA-seq (e.g., Rhapsody) cartridge along with a large number of scS beads can help eliminate / minimize crosstalk between cells and crosstalk due to cytokines present in the supernatant. In some embodiments provided herein, any background, if present, can be detected by the presence of capture Ab-seq PCR product in wells without cells.

[0156] The disclosed compositions and methods support drug development efforts, where the effects of small molecules or therapeutic agents can be evaluated in multiple patient samples. Each patient sample can be identified by a sample tag sequence, and cells can be dispensed into scRNA-seq (e.g., Rhapsody) wells to obtain single cells and treat them with the small molecule of interest. 5,000–10,000 single cells can be analyzed in a single cartridge, supporting the evaluation of effects in 50–100 cells from 100 patients. Furthermore, samples from multiple scRNA-seq (e.g., Rhapsody) cartridges can be combined into a single RNA-seq run.

[0157] The disclosed compositions and methods can be used with existing scRNA-seq cartridges (e.g., Rhapsody cartridges) to isolate single cells, enabling an ultrasensitive and quantitative method for evaluating secretome information along with RNA and surface proteins using scRNA-seq workflow elements. With current single-cell methods for detecting secreted factors, there is high background and crosstalk between cytokine-secreting cells and bystander cells. Furthermore, the number of secreted proteins that can be detected by currently available methods is limited to a small number of secreted antigens, or the methods are low-throughput and very time-consuming. Including single cells with a large number of scS beads in an scRNA-seq cartridge (e.g., Rhapsody cartridge) can help eliminate / minimize crosstalk between cells and crosstalk due to cytokines present in the supernatant. In some embodiments, any background (if present) can be detected by the presence of capture Ab-seq PCR products in wells without cells. The disclosed scRNA-seq (e.g., Rhapsody) workflow enables high-throughput analysis and the combination of cells from a large number of samples, donors, etc.

[0158] Characterizing cells at the single-cell level based on their secretion can be highly important for identifying optimal polyfunctional antigen-specific CD4+ and CD8+ T cells and associated T cell receptor (TCR) sequences. The methods provided herein allow for the identification of TCR sequences associated with functionally effective cells. Furthermore, the efficacy of CAR-T cells depends on their ability to react with antigens by secreting immune factors and their long-term fate. This depends on numerous factors, such as donor cells, treatment methods, the type of chimeric antigen receptor and its subdomains, endogenous TCRs, and others. The methods and compositions provided herein can be used to improve the efficacy of CAR-T cells.

[0159] The disclosed compositions and methods can use one or more technical principles of ELISA / ELISPOT, CBA array, IMag, and Ab-seq and can be integrated with scRNA-seq (e.g., Rhapsody) workflows to evaluate single cells. In some embodiments, beads conjugated with antibodies that detect intracellular proteins are provided to enable sensitive, high-throughput estimation of proteins, phosphorylated forms, and other conjugated states at the single-cell level. Some embodiments of the compositions and methods provided herein use a cell lysis buffer that does not affect cellular proteins. The disclosed compositions and methods can address the current need for sensitive methods for detecting secreted factors along with RNA and surface proteins in scRNA-seq (e.g., Rhapsody) workflows. Characterizing cells based on their secreted / released proteins can be highly important for distinguishing polyfunctional antigen-specific CD4+ and CD8+ T cells and associated T cell receptor (TCR) sequences, transcriptome profiles, and for assessing response and immunotherapy outcomes.

[0160] Systems, methods, compositions, and kits for measuring secreted analytes from cells are described in U.S. Patent Application Publication No. 20210222244, entitled "METHODS AND COMPOSITIONS FOR SINGLE CELL SECRETOMICS," the contents of which are incorporated herein by reference in their entirety. The systems, methods, compositions, and kits provided herein can, in some embodiments, be used with the systems, methods, compositions, and kits for secretome analysis described in U.S. Patent Application Publication No. 20220178909 and U.S. Patent Application Publication No. 20220178909, the contents of which are incorporated herein by reference in their entirety.

[0161] In some embodiments of the methods and compositions provided herein, DNA cellular component binding reagent-specific oligonucleotides (e.g., antibody oligonucleotides) hybridize to and extend oligonucleotide barcodes, enabling separate but parallel workflows for protein quantification and mRNA quantification from the same bead, as described in U.S. Patent No. 1,164,949,7 B2, the contents of which are incorporated herein by reference in their entirety. Some embodiments of the methods and compositions provided herein use the separate but parallel workflow concept described in U.S. Patent No. 1,714,727, for example, in some embodiments, secreted analyte binding reagent-specific oligonucleotides (e.g., antibody oligonucleotides) hybridize to and extend oligonucleotide barcodes, enabling separate but parallel workflows for secreted analyte quantification and mRNA quantification from the same bead.

[0162] In some embodiments of the methods and compositions provided herein, the oligonucleotide barcode comprises a cleavage region (e.g., comprising one or more cleavage sites such as non-canonical nucleotides (e.g., deoxyuridine) or restriction enzyme recognition sequences) as described in US20210214770A1, the contents of which are incorporated herein by reference in their entirety.

[0163] 4A-4G show schematic diagrams of non-limiting exemplary workflows for measuring the copy number of one or more secreted analytes secreted by single cells. The workflow may include step 400a of distributing first solid supports (404a, 404b, 404c, 404d) into compartments 402 of a plurality of compartments. Each first solid support may include a plurality of capture reagents (406a, 406b, 406c, 406d) capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell. The workflow may include step 400b of contacting a plurality of single cells (401a and 401b) (e.g., T cells, B cells, tumor cells, bone marrow cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof) with a plurality of second solid supports 403 to generate one or more single cells associated with the second solid supports. The single cell 401a may include a surface cellular target 408. The single cell 401b may lack a surface cell target 408. The second solid support 403 may include an isolation reagent 406 capable of specifically binding to the surface cell target 408. The cell 401a may include a secretory vesicle 410 containing unreleased secreted analytes 412a, 412b, 412c, and 412d. The secreted analytes 412a, 412b, 412c, and 412d may be different secreted analytes. The cell 401a may be capable of secreting the secreted analytes 412a, 412b, 412c, and 412d. The cell 401a may include copies 418 of a nucleic acid target. The workflow may include isolating 400c a single cell (e.g., a cell of interest) associated with the second solid support. The workflow may include distributing 400d the cell to compartments 402 of the plurality of compartments. A compartment of the plurality of compartments comprises one or more first solid supports of the first plurality of first solid supports and one or more single cells. The method may include an incubation step 400e to allow secretion of secreted analytes 412a, 412b, 412c, and 412d and capture by capture reagents 406a, 406b, 406c, and 406d.The workflow may include a step 400f of contacting the compartments with secreted analyte binding reagents (418a, 418b, 418c, and 418d), each capable of specifically binding to the capture reagent-bound secreted analyte. Each of the multiple secreted analyte binding reagents (418a, 418b, 418c, and 418d) may include a secreted analyte binding reagent-specific oligonucleotide (420a, 420b, 420c, and 420d) that includes a unique analyte identifier sequence for the secreted analyte binding reagent. The workflow may include an incubation step 400g (to allow binding of the secreted analyte binding reagent to the capture reagent-bound secreted analyte). The workflow may include a washing step 400h to remove unbound secreted analyte binding reagent. The workflow may include applying a magnetic field to the multiple compartments during the washing step. The solid supports provided herein (e.g., first solid support, second solid support, third solid support) may comprise a magnetic material and therefore may remain in the compartment during the washing step. The workflow may include contacting 400i a third solid support 422. A compartment of the plurality of compartments may include a single third solid support 422. The third solid support 422 may include multiple oligonucleotide barcodes 424. The oligonucleotide barcodes 424 may include a first molecular label and / or a cellular label. The workflow may include contacting the oligonucleotide barcodes 424 with secreted analyte-binding reagent-specific oligonucleotides 420 for hybridization. The workflow may include barcoding, library preparation, and / or sequencing 400j described herein. For example, the workflow may include extending an oligonucleotide barcode 424 hybridized to a secreted analyte-binding reagent-specific oligonucleotide 420 to generate a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each of which includes a sequence complementary to at least a portion of the unique analyte identifier sequence and a first molecular label.The method may include obtaining sequence information of the barcoded secreted analyte-binding reagent-specific oligonucleotide or its product to determine the number of copies of the secreted analyte 412 secreted by each of one or more single cells 401 a. The workflow may include performing the steps with a plurality of cells (e.g., in bulk).

[0164] 5 shows a non-limiting exemplary design of a secreted analyte-binding reagent-specific oligonucleotide (antibody oligonucleotide as shown herein) associated with a secreted analyte-binding reagent (antibody as shown herein). The secreted analyte-binding reagent-specific oligonucleotide 504 may be associated with the secreted analyte-binding reagent 502 through a linker 516. The secreted analyte-binding reagent-specific oligonucleotide 504 can be detached from the secreted analyte-binding reagent 502 using chemical, optical, or other means. The secreted analyte-binding reagent-specific oligonucleotide 504 may be an mRNA mimic. The secreted analyte-binding reagent-specific oligonucleotide 504 may include a second universal sequence 506 (e.g., a primer adapter), a second molecular label 508 (e.g., a unique molecular label sequence), an antibody barcode 510 (e.g., a unique analyte identifier sequence), an alignment sequence 512, and a poly(A) tail 514.

[0165] In some embodiments, a method is provided for measuring the copy number of a secreted analyte secreted by a single cell, the method comprising the steps of: distributing a plurality of first solid supports and one or more single cells into a plurality of compartments, each compartment comprising one or more first solid supports of a first plurality of first solid supports and one or more single cells, wherein the one or more single cells are capable of secreting a plurality of secreted analytes, each first solid support comprising a plurality of capture reagents capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell; and contacting the one or more first solid supports with a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte to which the capture reagent is bound, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent. The method may include the steps of: contacting a plurality of oligonucleotide barcodes with secreted analyte-binding reagent-specific oligonucleotides for hybridization, wherein the oligonucleotide barcodes each comprise a first molecular label; extending the plurality of oligonucleotide barcodes hybridized to the secreted analyte-binding reagent-specific oligonucleotides to generate a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each barcode comprising a sequence complementary to at least a portion of the unique analyte identifier sequence and the first molecular label; and obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or their products to determine the copy number of at least one secreted analyte secreted by each of the one or more single cells. In some embodiments, the method provided herein includes isolating complexes from the plurality of compartments. The complexes may comprise the third solid support, the first solid support, and / or the secreted analyte-binding reagent. Isolating the complexes may include the use of a magnetic field.

[0166] In some embodiments, methods are provided for determining the copy number of a secreted analyte secreted by a single cell and the copy number of a nucleic acid target in a single cell.The method includes the steps of: distributing a plurality of first solid supports and one or more single cells into a plurality of compartments, each compartment comprising one or more first solid supports of a first plurality of first solid supports and one or more single cells, wherein the one or more single cells comprise a copy of a nucleic acid target, and the one or more single cells are capable of secreting a plurality of secreted analytes, each first solid support comprising a plurality of capture reagents capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell; contacting the one or more first solid supports with a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte to which the capture reagent is bound, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent; and contacting a plurality of oligonucleotide barcodes for hybridization with the secreted analyte binding reagent-specific oligonucleotides and the copies of the nucleic acid target. extending the plurality of oligonucleotide barcodes hybridized to copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules, each comprising the first molecular label and a sequence complementary to at least a portion of the nucleic acid target; extending the plurality of oligonucleotide barcodes hybridized to the secreted analyte-binding reagent-specific oligonucleotides to generate a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each comprising the first molecular label and a sequence complementary to at least a portion of the unique analyte identifier sequence; obtaining sequence information of the plurality of barcoded nucleic acid molecules or products thereof to determine the copy number of the nucleic acid target in each of the one or more single cells; and obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one secreted analyte secreted by each of the one or more single cells.

[0167] In some embodiments, the one or more single cells comprise one or more single cells associated with a second solid support, and the method may include, prior to the distributing step, contacting the population of single cells with a plurality of second solid supports to generate one or more single cells associated with the second solid support, wherein the one or more single cells comprise a surface cell target, and each second solid support comprises a plurality of isolation reagents, each of the plurality of isolation reagents being capable of specifically binding to the surface cell target, and the method may include removing a single cell of the population of single cells that is not associated with the second solid support. In some embodiments, the one or more single cells are one or more cell types of interest, and the cell types of interest may comprise surface cell targets to which the isolation reagents of the second solid support can bind. In some embodiments, the one or more cell types of interest comprise hemogenic endothelial cells, hematopoietic stem and progenitor cells (HSCs), hematopoietic multipotent progenitor cells (MPPs), pre-T cell precursors, pre-K cell precursors, T cell precursors, NK cell precursors, T cells, NK cells, NKT cells, B cells, macrophages, neutrophils, CD4 cells, CD8 cells, naive T cells, stem cell-like memory T cells, central memory T cells, double-negative T cells, effector memory T cells, effector T cells, Th0 (Th0) cells, Tc0 (Tc0) cells, Th1 (Thl) cells, Tc1 (Tel) cells, Th2 cells, Tc2 cells, Th17 (Thl7) cells, Th22 cells, gamma / delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic pluripotent stem cells, or any combination thereof. In some embodiments, the one or more cell types of interest comprise one or more immune cell types, optionally naive CD4 T cells, effector memory CD4 T cells, naive CD8 T cells, effector memory CD8 T cells, naive CD4 Treg cells, effector memory CD4 Treg cells, naive B cells, memory B cells, CD16 DCs, plasmacytoid DCs, or any combination thereof.

[0168] The method may include, after contacting one or more first solid supports with a plurality of secreted analyte-binding reagents, removing one or more secreted analyte-binding reagents that are not in contact with one or more first solid supports of the plurality of secreted analyte-binding reagents. In some embodiments, removing one or more secreted analyte-binding reagents that are not in contact with one or more first solid supports comprises removing one or more secreted analyte-binding reagents that are not in contact with at least one of the respective capture reagent-bound secreted analytes. In some embodiments, the first solid support and / or the second solid support comprise a magnetic material, optionally a ferromagnetic material. In some embodiments, the removing step may include applying a magnetic field to the plurality of compartments, and single cells associated with the second solid support and one or more first solid supports may be able to remain in the compartments when the magnetic field is applied. In some embodiments, the method comprises one or more incubation steps for a period of time, which may be about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, or about 240 minutes, and further, said incubation may occur after (i) contacting the single cell with a first solid support and / or (ii) contacting the one or more first solid supports with a plurality of secreted analyte binding reagents.

[0169] In some embodiments, a compartment of the plurality of compartments comprises from about 2 to about 20 first solid supports, optionally about 8 first solid supports, which may be the same or different, and further, each first solid support may comprise a single type of capture reagent and / or different types of capture reagents. In some embodiments, the first solid support and / or second solid support is less than about 15 μm, less than about 12 μm, less than about 10 μm, less than about 9 μm, less than about 8 μm, less than about 7 μm, less than about 6 μm, less than about 5 μm, less than about 4 μm, less than about 3 μm, less than about 2 μm, less than about 1 μm, less than about 0.8 μm, less than about 0.6 μm, less than about 0.5 μm, less than about 0.4 μm, less than about 0.2 μm, less than about 0.1 μm, or less than about 0.01 μm.

[0170] Some embodiments disclosed herein provide a plurality of compositions, each comprising a secreted analyte-binding reagent (e.g., a protein-binding reagent). The secreted analyte-binding reagent may be conjugated to an oligonucleotide, where the oligonucleotide comprises a unique analyte identifier for the conjugated secreted analyte-binding reagent. The unique analyte identifier may be, for example, a nucleotide sequence having any suitable length, for example, from about 4 nucleotides to about 200 nucleotides. In some embodiments, the unique analyte identifier is a nucleotide sequence from 25 nucleotides to about 45 nucleotides in length. In some embodiments, the unique analyte identifier may have a length that is 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 200 nucleotides, is about, is less than, is greater than, or is within a range between any two of the above values.

[0171] In some embodiments, the unique analyte identifiers are selected from a diverse set of unique analyte identifiers, which may include 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or range between any two of these values, or may include approximately these values ​​or such numbers or ranges of unique analyte identifiers. A diverse set of unique analyte identifiers may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000 different unique analyte identifiers, or may include up to these values ​​of different unique analyte identifiers. In some embodiments, the set of unique analyte identifiers are designed to have minimal sequence homology to the DNA or RNA sequences of the sample being analyzed. In some embodiments, the sequences of the set of unique analyte identifiers differ from each other or their complements by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, or a number or range between any two of these values, or by approximately these values ​​or such number or range. In some embodiments, the sequences of a set of unique analyte identifiers differ from each other or their complements by at least, or by up to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, hi some embodiments, the sequences of a set of unique analyte identifiers differ from each other or their complements by at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, or more.

[0172] Any suitable secreted analyte binding reagent, isolation reagent, and capture reagent, such as a protein binding reagent, an antibody or fragment thereof, an aptamer, a small molecule, a ligand, a peptide, an oligonucleotide, etc., or any combination thereof, is contemplated in the present disclosure. The secreted analyte binding reagent, isolation reagent, and / or capture reagent can be a polyclonal antibody, a monoclonal antibody, a recombinant antibody, a single-chain antibody (sc-Ab) or a fragment thereof, such as Fab, Fv, etc. In some embodiments, the plurality of secreted analyte binding reagents can include 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or range between any two of these values, or can include approximately these values ​​or such numbers or ranges of different secreted analyte binding reagents. In some embodiments, the plurality of secreted analyte binding reagents may include at least, or up to, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000 different secreted analyte binding reagents. In some embodiments, the plurality of isolated reagents may include, or may include about, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or range between any two of these values. In some embodiments, the plurality of isolation reagents may include at least, or may include up to, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000 different isolation reagents.In some embodiments, the plurality of capture reagents may include 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, or a number or range between any two of these values. In some embodiments, the plurality of capture reagents may include at least or up to 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, or 5000 different capture reagents.

[0173] The oligonucleotide may be conjugated to the secreted analyte-binding reagent by various mechanisms. In some embodiments, the oligonucleotide may be covalently conjugated to the secreted analyte-binding reagent. In some embodiments, the oligonucleotide may be non-covalently conjugated to the secreted analyte-binding reagent. In some embodiments, the oligonucleotide is conjugated to the secreted analyte-binding reagent through a linker. The linker may be, for example, cleavable or detachable from the secreted analyte-binding reagent and / or the oligonucleotide. In some embodiments, the linker may include a chemical group that reversibly binds the oligonucleotide to the secreted analyte-binding reagent. The chemical group may be conjugated to the linker through, for example, an amine group. In some embodiments, the linker may include a chemical group that forms a stable bond with another chemical group conjugated to the secreted analyte-binding reagent. For example, the chemical group may be a group cleavable by UV light, a disulfide bond, streptavidin, biotin, an amine, etc. In some embodiments, the chemical group may be conjugated to the secreted analyte-binding reagent through a primary amine on an amino acid, such as lysine, or through the N-terminus. To conjugate an oligonucleotide to a secreted analyte-binding reagent, commercially available conjugation kits, such as the Protein-Oligo Conjugation Kit (Solulink, Inc., San Diego, California), the Thunder-Link® Oligo Conjugation System (Innova Biosciences, Cambridge, United Kingdom), and the like, can be used.

[0174] The oligonucleotide may be conjugated to any suitable site on the secreted analyte binding reagent (e.g., a protein binding reagent) so long as it does not interfere with the specific binding of the secreted analyte binding reagent to its secreted analyte. In some embodiments, the secreted analyte binding reagent is a protein, such as an antibody. In some embodiments, the secreted analyte binding reagent is not an antibody. In some embodiments, the oligonucleotide is conjugated anywhere other than the antigen binding site, e.g., to the Fc region, C H 1 domain, CH 2 domains, C H 3 domains, C L The oligonucleotide may be conjugated to an antibody, such as a domain. Methods for conjugating an oligonucleotide to a binding reagent (e.g., an antibody) have been previously disclosed, for example, in U.S. Patent No. 6,531,283, the contents of which are hereby expressly incorporated by reference in their entirety. The stoichiometry of the oligonucleotide and the secreted analyte-binding reagent can be varied. To increase the sensitivity of detection of secreted analyte-binding reagent-specific oligonucleotides in sequencing, it may be advantageous to increase the ratio of oligonucleotide to secreted analyte-binding reagent in the conjugation. In some embodiments, each secreted analyte-binding reagent may be conjugated to a single oligonucleotide molecule. In some embodiments, each secreted analyte-binding reagent may be conjugated to two or more oligonucleotide molecules, for example, at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, or a number or range between any two of these values, or up to these values ​​or a number or range of oligonucleotide molecules, each of which contains the same or different unique analyte identifiers. In some embodiments, each secreted analyte binding reagent may be conjugated to two or more oligonucleotide molecules, for example, at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000 oligonucleotide molecules, or up to these values ​​of oligonucleotide molecules, each of which comprises the same or different unique analyte identifier.

[0175] In some embodiments, the plurality of secreted analyte binding reagents are capable of specifically binding to a plurality of secreted analytes in a sample, such as a single cell, a plurality of cells, a tissue sample, a tumor sample, a blood sample, etc. In some embodiments, the plurality of secreted analytes may include 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10,000, or a number or range between any two of these values, or may include approximately these values ​​or such numbers or ranges of different secreted analytes. In some embodiments, the plurality of secreted analytes may include at least, or may include up to, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 1000, 10,000 different secreted analytes.

[0176] In some embodiments, the secreted analyte binding reagent-specific oligonucleotide is about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 53 , 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 nucleotides in length, or a number or range between any two of these values.In some embodiments, the secreted analyte binding reagent-specific oligonucleotide is at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 128, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 , 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, or a nucleotide sequence up to these values ​​in length.

[0177] Oligonucleotide-conjugated antibodies Some embodiments disclosed herein provide a plurality of compositions each comprising a cellular component binding reagent (e.g., a protein binding reagent) conjugated to an oligonucleotide, the plurality of compositions comprising a unique identifier for the cellular component binding reagent to which the oligonucleotide is conjugated. Cellular component binding reagents (e.g., barcoded antibodies, etc.) and their uses (e.g., cellular sample indexing) are described in U.S. Patent Application Publication Nos. 2018 / 0088112 and 2018 / 0346970, the contents of each of which are incorporated herein by reference in their entireties. Some embodiments provided herein provide a secreted analyte binding reagent capable of specifically binding to a secreted analyte. The secreted analyte binding reagent may comprise a secreted analyte-binding reagent-specific oligonucleotide. Some embodiments provide a method for simultaneous quantitative analysis of multiple cellular component targets (e.g., protein targets) and copies of a secreted analyte secreted by a single cell. The methods and systems described herein can be used with methods and systems that use antibodies associated with (e.g., bound or conjugated to) oligonucleotides (also referred to herein as AbOs or AbOligos). Embodiments using AbOs to determine protein expression profiles in single cells and track sample origin are described in U.S. Patent Application Publication Nos. 2018 / 0088112 and 2018 / 0346970, the contents of each of which are incorporated herein by reference in their entireties.

[0178] Unique molecular beacon sequences In some embodiments, the methods and compositions provided herein include an oligonucleotide associated with a cellular component binding reagent (e.g., an antibody oligonucleotide ("AbOligo" or "AbO"), a binding reagent oligonucleotide, a cellular component binding reagent-specific oligonucleotide, a sample-indexing oligonucleotide), as described in U.S. Patent Application Publication No. 16 / 747,737, filed January 21, 2020, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the oligonucleotide associated with a cellular component binding reagent (e.g., an antibody oligonucleotide ("AbOligo" or "AbO"), a binding reagent oligonucleotide, a secreted analyte-binding reagent-specific oligonucleotide, a cellular component binding reagent-specific oligonucleotide, a sample-indexing oligonucleotide) comprises a unique molecular label sequence (also referred to as a molecular index (MI), a "molecular barcode," or a unique molecular identifier (UMI)). In some embodiments, binding reagent oligonucleotide species comprising molecular barcodes described herein reduce bias by increasing sensitivity, decreasing relative standard error, or increasing sensitivity and / or decreasing standard error. Molecular barcodes can contain unique sequences, and thus, when multiple sample nucleic acids (which can be the same and / or different from one another) are associated one-to-one with molecular barcodes, the different sample nucleic acids can be distinguished from one another by the molecular barcodes. Thus, even if a sample contains two nucleic acids with the same sequence, each of these two nucleic acids can be labeled with a different molecular barcode, and thus the nucleic acids in a population can be quantified, even after amplification.A molecular barcode may comprise at least 5 nucleotides, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 5 0 nucleotides (ranges between any two of the listed values, e.g., 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10 , 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides). In some embodiments, the nucleic acid sequence of the molecular barcode comprises a unique sequence, e.g., such that each unique oligonucleotide species in the composition comprises a different molecular barcode. In some embodiments, three or more unique oligonucleotide species may comprise the same molecular barcode but are still different from one another. For example, if a unique oligonucleotide species comprises a sample barcode, each unique oligonucleotide species with a particular sample barcode may comprise a different molecular barcode. In some embodiments, a composition comprising unique oligonucleotide species comprises a molecular barcode diversity of at least 1000 different molecular barcodes and therefore at least 1000 unique oligonucleotide species.In some embodiments, a composition comprising unique oligonucleotide species comprises a molecular barcode diversity of at least 6,500 different molecular barcodes and therefore at least 6,500 unique oligonucleotide species. In some embodiments, a composition comprising unique oligonucleotide species comprises a molecular barcode diversity of at least 65,000 different molecular barcodes and therefore at least 65,000 unique oligonucleotide species.

[0179] In some embodiments, the unique molecular label sequence is located 5' to the unique identifier sequence, with no intervening sequence between the unique molecular label sequence and the unique identifier sequence. In some embodiments, the unique molecular label sequence is located 5' to the spacer that is located 5' to the unique identifier sequence, with the spacer therefore being between the unique molecular label sequence and the unique identifier sequence. In some embodiments, the unique identifier sequence is located 5' to the unique molecular label sequence, with no intervening sequence between the unique identifier sequence and the unique molecular label sequence. In some embodiments, the unique identifier sequence is located 5' to the spacer that is located 5' to the unique molecular label sequence, with the spacer therefore being between the unique identifier sequence and the unique molecular label sequence.

[0180] A unique molecular label sequence may be at least 3 nucleotides, e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 nucleotides (a range between any two of the listed values, e.g., 3-50 ... ~45, 3~40, 3~35, 3~30, 3~25, 3~20, 3~15, 3~14, 3~13, 3~12, 3~11, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~5, 3~4, 4~50, 4~45, 4~40, 4~35, 4~30, 4~25, 4~20, 4~15, 4~14, 4~13, 4~12, 4~11, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~50, 5~45, 5~40, 5~35, 5~30, 5~25, 5~20, 5~15, 5 ~14, 5~13, 5~12, 5~11, 5~10, 5~9, 5~8, 5~7, 5~6, 6~50, 6~45, 6~40, 6~35, 6~30, 6~25, 6~20, 6~15, 6~14, 6~13, 6~12, 6~11, 6~10, 6~9, 6~8, 6~7, 7~50, 7~45, 7~40, 7~35, 7~30, 7~25, 7~20, 7~15, 7~14, 7~13, 7~12, 7~11, 7~10, 7~9, 7~8, 8~50, 8~45, 8~40, 8~35 , 8-30, 8-25, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-50, 9-45, 9-40, 9-35, 9-30, 9-25, 9-20, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 10-14, 10-13, 10-12, or 10-11 nucleotides). In some embodiments, the unique molecular label sequence is 2-20 nucleotides in length.

[0181] In some embodiments, the unique molecular beacon sequence of the binding reagent oligonucleotide comprises at least three repeats of the doublet "VN" and / or "NV" (where each "V" is either A, C, or G, and "N" is either A, G, C, or T), e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (including ranges between any two of the listed values) of the doublet "VN." Examples of multiple repeats of the doublet "VN" include VN, VNVN, VNVNVN, and VNVNVNVN. It is noted that while the formulas "VN" and "NV" describe constraints on base content, not all Vs or all Ns must be the same or different. For example, if the molecular barcodes of the unique oligonucleotide species in a composition include VNVNVN, one molecular barcode may include the sequence ACGGCA, another molecular barcode may include the sequence ATACAT, and another molecular barcode may include the sequence ATACAC. Note that any number of repeats of the doublet "VN" have a T content of 50% or less. In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise molecular barcodes that include at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (including ranges between any two of the listed values) of the doublet "VN" and / or "NV." In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise a molecular barcode that comprises at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (including ranges between any two of the listed values) of the doublets "VN" and / or "NV."In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 1000 unique oligonucleotide species comprise a molecular barcode that comprises at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (including ranges between any two of the enumerated values). In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise a molecular barcode that comprises at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (including ranges between any two of the enumerated values). In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise a molecular barcode that comprises at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (inclusive of ranges between any two of the enumerated values). In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 6500 unique oligonucleotide species comprise a molecular barcode that comprises at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (inclusive of ranges between any two of the enumerated values).In some embodiments, at least 95% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise a molecular barcode that comprises at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (inclusive of ranges between any two of the enumerated values). In some embodiments, at least 99% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise a molecular barcode that comprises at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (inclusive of ranges between any two of the enumerated values). In some embodiments, at least 99.9% of the unique oligonucleotide species of a composition comprising at least 65,000 unique oligonucleotide species comprise a molecular barcode that comprises at least three repeats of the doublet "VN" and / or "NV," e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats (including ranges between any two of the recited values). In some embodiments, the composition consists of, or consists essentially of, at least 1000, 6500, or 65,000 unique oligonucleotide species, each having a molecular barcode that comprises the sequence VNVNVN. In some embodiments, the composition consists of, or consists essentially of, at least 1000, 6500, or 65,000 unique oligonucleotide species, each having a molecular barcode that comprises the sequence VNVNVNVN. In some embodiments, at least 95%, 99%, or 99.9% of the barcode regions of the compositions described herein comprise at least three repeats of the doublets "VN" and / or "NV" described herein.In some embodiments, unique molecular beacon sequences comprising repeated doublets "VN" and / or "NV" can reduce bias and simultaneously provide a compromise between reducing bias and maintaining a relatively large number of available nucleotide sequences, thus allowing for relatively high diversity in relatively short sequences while still minimizing bias. In some embodiments, unique molecular beacon sequences comprising repeated doublets "VN" and / or "NV" can reduce bias by increasing sensitivity, reducing the relative standard error, or increasing sensitivity and reducing the standard error. In some embodiments, unique molecular beacon sequences comprising repeated doublets "VN" and / or "NV" improve informatics by serving as geomarkers. In some embodiments, the repeated doublets "VN" and / or "NV" described herein reduce the incidence of homopolymers within the unique molecular beacon sequence. In some embodiments, the repeated doublets "VN" and / or "NV" described herein separate homopolymers.

[0182] In some embodiments, the sample indexing oligonucleotide comprises a first molecular label sequence. In some embodiments, the first molecular label sequences of at least two sample indexing oligonucleotides are different, and the sample indexing sequences of at least two sample indexing oligonucleotides are identical. In some embodiments, the first molecular label sequences of at least two sample indexing oligonucleotides are different, and the sample indexing sequences of at least two sample indexing oligonucleotides are different. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises a second molecular label sequence. In some embodiments, the second molecular label sequences of at least two cellular component binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two cellular component binding reagent-specific oligonucleotides are identical. In some embodiments, the second molecular label sequences of at least two cellular component binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two cellular component binding reagent-specific oligonucleotides are different. In some embodiments, the number of unique second molecular label sequences associated with unique identifier sequences for cellular component binding reagents capable of specifically binding to at least one cellular component target in the sequencing data indicates the copy number of at least one cellular component target in one or more of the plurality of cells. In some embodiments, the combination (e.g., minimum, average, and maximum values) of (1) the number of unique first molecular label sequences associated with unique identifier sequences for cellular component binding reagents capable of specifically binding to at least one cellular component target in the sequencing data and (2) the number of unique second molecular label sequences associated with unique identifier sequences for cellular component binding reagents capable of specifically binding to at least one cellular component target in the sequencing data indicates the copy number of at least one cellular component target in one or more of the plurality of cells.

[0183] Alignment sequence In some embodiments, the binding reagent oligonucleotide comprises an alignment sequence (e.g., alignment sequence 825bb) adjacent to the poly(dA) region. The alignment sequence can be one or more nucleotides in length. The alignment sequence can be two nucleotides in length. The alignment sequence can comprise guanine, cytosine, thymine, uracil, or a combination thereof. The alignment sequence can comprise a poly(dT) region, a poly(dG) region, a poly(dC) region, a poly(dU) region, or a combination thereof. In some embodiments, the alignment sequence is 5' to the poly(dA) region. Advantageously, in some embodiments, the presence of the alignment sequence allows the poly(A) tails of each of the binding reagent oligonucleotides to have the same length, resulting in greater uniformity in performance. In some embodiments, the percentage of binding reagent oligonucleotides having the same length of poly(dA) region within a plurality of binding reagent oligonucleotides, each comprising an alignment sequence, can be 80%, 90%, 91%, 93%, 95%, 97%, 99.9%, 99.9%, 99.99%, or 100%, or a number or range between any two of these values, or can be approximately these values ​​or such numbers or ranges. In some embodiments, the percentage of binding reagent oligonucleotides having the same length of poly(dA) region within a plurality of binding reagent oligonucleotides, each comprising an alignment sequence, can be at least 80%, 90%, 91%, 93%, 95%, 97%, 99.9%, 99.9%, 99.99%, or 100%.

[0184] The length of the alignment sequence may vary in different implementations. In some embodiments, the length of the alignment sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or range between any two of these values, or may be approximately any such value or such number or range. In some embodiments, the length of the alignment sequence is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, The number of guanines, cytosines, thymines, or uracils in the aligned sequences may be, or may be up to, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. The number of guanines, cytosines, thymines, or uracils in the aligned sequences may vary in different implementations.The number of guanines, cytosines, thymines, or uracils is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or a number or range between any two of these values, or may be approximately any such value or such number or range. The number of guanines, cytosines, thymines, or uracils is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, The number of oligonucleotides may be, or may be up to, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. In some embodiments, the sample-indexing oligonucleotide comprises an alignment sequence. In some embodiments, the cellular component-binding reagent-specific oligonucleotide and / or the secreted analyte-binding reagent-specific oligonucleotide comprises an alignment sequence.

[0185] Linker The binding reagent oligonucleotide (e.g., a secreted analyte-binding reagent-specific oligonucleotide) may be conjugated to the cellular component binding reagent by various mechanisms. In some embodiments, the binding reagent oligonucleotide may be covalently conjugated to the cellular component binding reagent. In some embodiments, the binding reagent oligonucleotide may be non-covalently conjugated to the cellular component binding reagent. In some embodiments, the binding reagent oligonucleotide is conjugated to the cellular component binding reagent through a linker. In some embodiments, the binding reagent oligonucleotide may comprise a linker. The linker may comprise a chemical group. The chemical group may be reversibly or irreversibly bound to the cellular component binding reagent molecule. The chemical group may be selected from a group cleavable by UV light, a disulfide bond, streptavidin, biotin, an amine, and combinations thereof. The linker may comprise a carbon chain. The carbon chain may comprise, for example, 5 to 50 carbon atoms. The carbon chain may have a different number of carbon atoms in different embodiments. In some embodiments, the number of carbon atoms in the carbon chain can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or a number or range between any two of these values, or can be about these values ​​or such numbers or ranges. In some embodiments, the number of carbon atoms in the carbon chain can be at least, or can be up to, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the carbon chain contains between 2 and 30 carbons. In some embodiments, the carbon chain contains 12 carbons. In some embodiments, the amino modifier used in the binding reagent oligonucleotide can be conjugated to a cellular component binding reagent.In some embodiments, the linker comprises a 5' amino modifier C6 (5AmMC6). In some embodiments, the linker comprises a 5' amino modifier C12 (5AmMC12). In some embodiments, the linker comprises a derivative of 5AmMC12. In some embodiments, the longer the linker, the higher the conjugation efficiency achieved. In some embodiments, the longer the linker, the higher the pre-conjugation modification efficiency achieved. In some embodiments, increasing the distance between the functional amine and the DNA sequence results in higher conjugation efficiency. In some embodiments, increasing the distance between the functional amine and the DNA sequence results in higher pre-conjugation modification efficiency. In some embodiments, using 5AmMC12 as a linker results in higher modification efficiency (pre-conjugation) than using 5AmMC6 as a linker. In some embodiments, using 5AmMC12 as a linker results in higher conjugation efficiency than using 5AmMC6 as a linker. In some embodiments, the sample-indexing oligonucleotide is associated with the cellular component binding reagent through a linker. In some embodiments, the cellular component binding reagent-specific oligonucleotide and / or the secreted analyte-binding reagent-specific oligonucleotide is associated with the cellular component binding reagent through a linker.

[0186] Antibody-specific barcode sequence In some embodiments, improvements to the design of unique identifier sequences (e.g., antibody-specific barcode sequences) of binding reagent oligonucleotides (e.g., secreted analyte-binding reagent-specific oligonucleotides) are disclosed herein. In some embodiments, the unique identifier sequences (e.g., sample-indexing sequences of cellular component-binding reagent-specific oligonucleotides, unique analyte identifier sequences, unique identifier sequences) are designed to have a Hamming distance greater than 3. In some embodiments, the Hamming distance of the unique identifier sequences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a number or range between any two of these values, or can be approximately these values ​​or such a number or range. In some embodiments, the unique identifier sequences have a GC content within the range of 40% to 60% and are free of predicted secondary structure (e.g., hairpins). In some embodiments, the unique identifier sequences do not include any sequences predicted in silico to bind to mouse and / or human transcripts. In some embodiments, the unique identifier sequences do not include any sequences predicted in silico to bind to Rhapsody™ and / or SCMK system primers. In some embodiments, the unique identifier sequence does not include homopolymers.

[0187] Primer Adapter In some embodiments, the binding reagent oligonucleotide (e.g., secreted analyte-binding reagent-specific oligonucleotide) comprises a primer adapter. In some embodiments, the primer adapter comprises the sequence of a first universal primer, its complementary sequence, a subsequence thereof, or a combination thereof. In some embodiments, the first universal primer comprises an amplification primer, its complementary sequence, a subsequence thereof, or a combination thereof. In some embodiments, the first universal primer comprises a sequencing primer, its complementary sequence, a subsequence thereof, or a combination thereof. In some embodiments, the sequencing primer comprises an Illumina sequencing primer. In some embodiments, the sequencing primer comprises a portion of an Illumina sequencing primer. In some embodiments, the sequencing primer comprises a P7 sequencing primer or a portion of a P7 sequencing primer. In some embodiments, the primer adapter comprises an adapter for Illumina P7 or a partial adapter for Illumina P7. In some embodiments, the amplification primer is an Illumina P7 sequence or a subsequence thereof. In some embodiments, the sequencing primer is an Illumina R2 sequence or a subsequence thereof. In some embodiments, the first universal primer is 5 to 50 nucleotides in length.In some embodiments, the primer adapter comprises at least 5 nucleotides, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, or 50 nucleotides (ranges between any two of the listed values, e.g., 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-1 4, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-25, 8-20, 8-15, 8-14, 8-13, 8-12, 8-11, The nucleic acid sequence may comprise 8 to 10, 8 to 9, 9 to 50, 9 to 45, 9 to 40, 9 to 35, 9 to 30, 9 to 25, 9 to 20, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 10 to 14, 10 to 13, 10 to 12, or 10 to 11 nucleotides.The primer adapter may comprise a nucleic acid sequence of at least 5 nucleotides of the sequence of a first universal primer, an amplification primer, a sequencing primer, its complementary sequence, a subsequence, or a combination thereof, such as at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides (a range between any two of the listed values, e.g., 5-50, 5-45, 5-40, 5-35, 5-30, 5-25, 5-20, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-45, 6-40, 6-35, 6-30, 6-25, 6-20, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-50, 7-45, 7-40, 7-35, 7-30, 7-25, 7-20, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-50, 8-45, 8-40, 8-35, 8-30, 8-2 The nucleic acid sequence may comprise 5, 8 to 20, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 50, 9 to 45, 9 to 40, 9 to 35, 9 to 30, 9 to 25, 9 to 20, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 10 to 14, 10 to 13, 10 to 12, or 10 to 11 nucleotides.

[0188] A conventional amplification workflow for sequencing library preparation can use three rounds of PCR: a first round ("PCR1") using a target-specific primer and a primer for the universal Illumina sequencing primer 1 sequence; a second round ("PCR2") using nested target-specific primers and a primer for the universal Illumina sequencing primer 1 sequence flanked by Illumina sequencing primer 2 sequences; and a third round ("PCR3") adding Illumina P5 and P7 and a sample index. Advantageously, in some embodiments, the primer adapters disclosed herein enable a shorter and simpler workflow in library preparation compared to when the starting template (e.g., a sample-indexing oligonucleotide bound to a bead) does not have a primer adapter. In some embodiments, the primer adapter reduces the pre-sequencing PCR amplification of the template by one round (compared to when the template does not contain a primer adapter). In some embodiments, the primer adapter reduces the pre-sequencing PCR amplification of the template to one round (compared to when the template does not contain a primer adapter). In some embodiments, templates containing primer adapters do not require a PCR amplification step for Illumina sequencing adapter attachment, which requires pre-sequencing if the template does not contain a primer adapter. In some embodiments, the primer adapter sequence (or a subsequence thereof) is not part of the sequencing read of a sequencing template containing the primer adapter sequence, and therefore does not affect the read quality of the template containing the primer adapter. In some embodiments, the sequencing diversity of templates containing primer adapters is reduced compared to templates not containing primer adapters.

[0189] In some embodiments, the sample indexing oligonucleotide comprises a primer adapter. In some embodiments, replicating the sample indexing oligonucleotide, the barcoded sample indexing oligonucleotide, or a product thereof comprises generating a plurality of replicated sample indexing oligonucleotides using a first universal primer, a first primer comprising the sequence of the first universal primer, or a combination thereof. In some embodiments, replicating the sample indexing oligonucleotide, the barcoded sample indexing oligonucleotide, or a product thereof comprises generating a plurality of replicated sample indexing oligonucleotides using a first universal primer, a first primer comprising the sequence of the first universal primer, a second universal primer, a second primer comprising the sequence of the second universal primer, or a combination thereof. In some embodiments, the cellular component binding reagent-specific oligonucleotide and / or the secreted analyte binding reagent-specific oligonucleotide comprises a primer adapter, the sequence of the first universal primer, a complementary sequence thereof, a subsequence thereof, or a combination thereof.

[0190] solid support The first solid support, the second solid support, and / or the third solid support may comprise a synthetic particle or a planar surface. At least one of the plurality of oligonucleotide barcodes may be immobilized or partially immobilized on a synthetic particle. At least one of the plurality of oligonucleotide barcodes may be encapsulated or partially encapsulated within a synthetic particle. The synthetic particle may be disintegrable. The synthetic particle may comprise a bead. The beads may comprise sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; a material selected from polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; or disintegrable hydrogel particles.

[0191] In some embodiments, each of the plurality of oligonucleotide barcodes comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and combinations thereof. In some embodiments, each of the plurality of isolation reagents comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof. In some embodiments, each of the plurality of capture reagents comprises a linker functional group, the synthetic particle comprises a solid support functional group, and the support functional group and the linker functional group are associated with each other. The linker functional group and the support functional group can each be selected from C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof.

[0192] The secreted analyte binding reagent and the capture reagent may be capable of binding to distinct epitopes of the same secreted analyte. In some embodiments, one or more of the secreted analyte binding reagent, capture reagent, and isolation reagent comprise an antibody or fragment thereof. The antibody or fragment thereof may comprise a monoclonal antibody. The antibody or fragment thereof may comprise a Fab, Fab', F(ab'), Fv, scFv, dsFv, diabody, triabody, tetrabody, multispecific antibody formed from antibody fragments, single domain antibody (sdAb), single chain including complementary scFv (tandem scFv) or bispecific tandem scFv, Fv construct, disulfide-linked Fv, dual variable domain immunoglobulin (DVD-Ig) binding protein or nanobody, aptamer, affibody, affilin, affitin, affimer, alphabody, anticalin, avimer, DARPin, phinomer, Kunitz domain peptide, monobody, or any combination thereof. The capture reagent and / or isolation reagent may be conjugated to the first solid support and / or the second solid support via a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilic substitution reaction, a non-aldol carbonyl reaction, a carbon-carbon multiple bond addition, an oxidation reaction, a click reaction, or any combination thereof.

[0193] The at least one secreted analyte may comprise a lymphokine, an interleukin, a chemokine, or any combination thereof. The at least one secreted analyte may comprise a cytokine, a hormone, a molecular toxin, or any combination thereof. The at least one secreted analyte may comprise a nerve growth factor, a liver growth factor, a fibroblast growth factor, a vascular endothelial growth factor, a platelet-derived growth factor, a transforming growth factor, an osteoinductive factor, an interferon, a colony-stimulating factor, or any combination thereof. The at least one secreted analyte may be angiogenin, angiopoietin-1, angiopoietin-2, bNGF, cathepsin S, galectin-7, GCP-2, G-CSF, GM-CSF, PAI-1, PDGF-AA, PDGF-BB, PDGF-AB, PlGF, PlGF-2, SDF-1, Tie2, VEGF-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2, VEGF-R3, VEGF-6, angiopoietin, β-lactamase ... Etin-1, Angiopoietin-2, BLC, BRAK, CD186, ENA-78, Eotaxin-1, Eotaxin-2, Eotaxin-3, EpCAM, GDF-15, GM-CSF, GRO, HCC-4, I-309, IFN-γ, IL-1α, IL-1β, IL-1R4(ST2), IL-2, IL-2R, IL-3, IL-3Rα, IL-5, IL-6, IL-6R, IL-7, IL-8, IL-8RB, IL-11, I L-12, IL-12p40, IL-12p70, IL-13, IL-13R1, IL-13R2, IL-15, IL-15Rα, IL-16, IL-17, IL-17C, IL-17E, IL-17F, IL-17 R, IL-18, IL-18BPa, IL-18Rα, IL-20, IL-23, IL-27, IL-28, IL-31, IL-33, IP-10, I-TAC, LIF, LIX, LRP6, MadCAM-1, MC P-1, MCP-2, MCP-3, MCP-4, M-CSF, MIF, MIG, MIP-1 gamma, MIP-1α, MIP-1β, MIP-1δ, MIP-3α, MIP-3β, MPIF-1, PARC, PF4, RANTES, resistin, SCF, SCYB16, TACI, TARC, TSLP, TNF-α, TNF-R1, TRAIL-R4, TREM-1, activin A, amphiregulin, Axl, BDNF, BMP4,Cathepsin S, EGF, FGF-1, FGF-2, FGF-7, FGF-21, follistatin, galectin-7, Gas6, GDF-15, HB-EGF, HGF, IGFBP-1, IGFBP-3, LAP, NGF R, NrCAM, NT-3, NT-4, PAI-1, TGF-α, TGF-β, TGF-β3, TRAIL-R4, ADAMTS1, cathepsin S, FGF-2, follistatin, galectin-7, GCP-2, GDF-15, IGFBP-6, LIF, MMP-9, pro-MMP9, RANK, RANKL, RANTES, SDF-1, CXCR4, or any combination thereof.

[0194] Surface cell targets may include carbohydrates, lipids, proteins, extracellular proteins, cell surface proteins, cell markers, B cell receptors, T cell receptors, major histocompatibility complexes, tumor antigens, receptors, intracellular proteins, or any combination thereof. Surface cell targets may include carbohydrates, lipids, proteins, or any combination thereof. Surface cell targets may include CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD15u, CD15s, CD15su, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD D26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD4 3, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD6 6b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, C D83, CD84, CD85a, CD85d, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD9 9R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115,CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CD121b、CD122、CD123、CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CDw145、CD146、CD147、CD148、CDw149、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158e、CD158i、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CDw198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210、CDw210b、CD212、CD213a1、CD213a2、CD215、CD217a、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD236R、CD238、CD239、CD240CE、CD240DCE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD266、CD267、CD268、CD269、CD270、CD271、CD272、CD273、CD274、CD275、CD276、CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300e, C D301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD308, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD 322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, BCMA, HLA proteins, β2-microglobulin, or any combination thereof.

[0195] Simultaneous single-cell secretome, proteome, and transcriptome analysis In some embodiments, methods for quantitative analysis of the transcriptome and / or proteome of a single cell are provided. The methods and systems described herein can be used with methods and systems that use antibodies associated with (e.g., bound or conjugated to) oligonucleotides (also referred to herein as AbOs or AbOligos). Embodiments using AbOs to determine protein expression profiles in single cells and track sample origin are described in U.S. Patent Application No. 15 / 715,028, published as U.S. Patent Application Publication No. 2018 / 0088112, and U.S. Patent Application Publication No. 2018 / 0346970, the contents of each of which are incorporated herein by reference in their entireties. One or more single cells can contain multiple cellular component targets. The method may include the steps of contacting a plurality of cellular component binding reagents with one or more single cells, each of the plurality of cellular component binding reagents comprising a cellular component binding reagent-specific oligonucleotide including a unique identifier sequence for the cellular component binding reagent, the cellular component binding reagent being capable of specifically binding to at least one of a plurality of cellular component targets, contacting a plurality of oligonucleotide barcodes with the cellular component binding reagent-specific oligonucleotides for hybridization, each of the oligonucleotide barcodes including a molecular label and a first universal sequence, extending the plurality of oligonucleotide barcodes hybridized to the cellular component binding reagent-specific oligonucleotides to generate a plurality of barcoded cellular component binding reagent-specific oligonucleotides, each of the barcodes including a molecular label and a sequence complementary to at least a portion of the unique identifier sequence, and obtaining sequence information of the plurality of barcoded cellular component binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one cellular component target among the plurality of cellular component targets in each of the one or more single cells. The cellular component binding reagent-specific oligonucleotides may include a sequence complementary to a capture sequence configured to capture the cellular component binding reagent-specific oligonucleotide. The sequence complementary to the capture sequence may include a poly(dA) region.

[0196] In some embodiments, the plurality of barcoded cellular component binding reagent-specific oligonucleotides comprise a complement of the first universal sequence. The cellular component binding reagent-specific oligonucleotides may comprise a fourth universal sequence. In some embodiments, obtaining sequence information of the plurality of barcoded cellular component binding reagent-specific oligonucleotides or their products includes amplifying the plurality of barcoded cellular component binding reagent-specific oligonucleotides or their products using a primer capable of hybridizing to the first universal sequence or its complement and a primer capable of hybridizing to the fourth universal sequence or its complement to generate a plurality of amplified barcoded cellular component binding reagent-specific oligonucleotides, and obtaining sequencing data of the plurality of amplified barcoded cellular component binding reagent-specific oligonucleotides or their products. The obtaining sequence information may include binding a sequencing adapter to the plurality of barcoded cellular component binding reagent-specific oligonucleotides or their products. The method may include, after contacting the plurality of cellular component binding reagents with one or more single cells, removing one or more cellular component binding reagents from the plurality of cellular component binding reagents that have not contacted the one or more single cells. Removing one or more cellular component binding reagents that are not in contact with one or more single cells can include removing one or more cellular component binding reagents that are not in contact with at least one of the plurality of cellular component targets. The cellular component targets can include intracellular proteins, carbohydrates, lipids, proteins, extracellular proteins, cell surface proteins, cell markers, B cell receptors, T cell receptors, major histocompatibility complexes, tumor antigens, receptors, intracellular proteins, or any combination thereof. The cellular component targets can include housekeeping proteins, detection of which indicates the presence of a single cell in the compartment.

[0197] In some embodiments, a plurality of oligonucleotide barcodes are associated with a third solid support. A compartment of the plurality of compartments may comprise a single third solid support. A compartment may be a well or a droplet. Each oligonucleotide barcode may comprise a first universal sequence. The oligonucleotide barcode may comprise a target binding region comprising a capture sequence. The target binding region may comprise a poly(dT) region. The secreted analyte-binding reagent-specific oligonucleotide may comprise a sequence complementary to a capture sequence configured to capture the secreted analyte-binding reagent-specific oligonucleotide. The sequence complementary to the capture sequence may comprise a poly(dA) region. In some embodiments, the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides comprises a complement of the first universal sequence. The secreted analyte-binding reagent-specific oligonucleotide may comprise a second universal sequence.

[0198] In some embodiments, obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof comprises amplifying the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a second universal sequence or its complement to produce a plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides; and obtaining sequencing data of the plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof.

[0199] The secreted analyte-binding reagent-specific oligonucleotides may comprise a second molecular label. In some embodiments, at least 10 of the plurality of secreted analyte-binding reagent-specific oligonucleotides comprise different second molecular label sequences. In some embodiments, the second molecular label sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are identical. In some embodiments, the second molecular label sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different. In some embodiments, the number of unique first molecular label sequences associated with unique analyte identifier sequences for secreted analyte-binding reagents capable of specifically binding to at least one secreted analyte in the sequencing data indicates the copy number of at least one secreted analyte secreted by each of the one or more single cells. In some embodiments, the number of unique second molecular label sequences associated with unique analyte identifier sequences for secreted analyte-binding reagents capable of specifically binding to at least one secreted analyte in the sequencing data indicates the copy number of at least one secreted analyte secreted by each of the one or more single cells. In some embodiments, the method includes determining the copy number of at least one secreted analyte secreted by each of the one or more single cells based on the number of first molecular labels and / or second molecular labels having distinct sequences associated with a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof. In some embodiments, the method includes determining the copy number of at least one secreted analyte secreted by each of the one or more single cells based on the number of first molecular labels and / or second molecular labels having distinct sequences associated with a plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof. In some embodiments, obtaining the sequence information includes attaching sequencing adaptors to the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof.The secreted analyte-binding reagent-specific oligonucleotide may be configured to be releasable from the secreted analyte-binding reagent. The method may include dissociating the secreted analyte-binding reagent-specific oligonucleotide from the secreted analyte-binding reagent.

[0200] In some embodiments, the systems, methods, compositions, and kits provided herein can be used in conjunction with the systems, methods, compositions, and kits for flow surrogate assays described in International Publication No. WO2023172977, the contents of which are incorporated herein by reference in their entirety. For example, the present disclosure includes a plurality of detectable conjugates, each of which comprises a detectable moiety or a precursor thereof and a unique identifier-specific oligonucleotide comprising a sequence configured to bind to a unique analyte identifier sequence, wherein detectable conjugates capable of binding to the same unique analyte identifier sequence comprise the same detectable moiety or a precursor thereof, and detectable conjugates capable of binding to different unique analyte identifier sequences comprise different detectable moieties or precursors thereof. The methods provided herein may include using an instrument to measure the emission of the detectable moiety of each detectable conjugate as an indicator of the amount of secreted analyte-binding reagent bound to the secreted analyte to which the capture reagent is bound.

[0201] Determining the copy number of the nucleic acid target in each of the one or more single cells may include determining the copy number of the nucleic acid target in each of the one or more single cells based on the number of first molecular labels having distinct sequences, their complements, or a combination thereof, associated with a plurality of barcoded nucleic acid molecules or products thereof. The method may include contacting random primers with the plurality of barcoded nucleic acid molecules, each of the random primers comprising a third universal sequence or its complement, and extending the random primers hybridized to the plurality of barcoded nucleic acid molecules to generate a plurality of extension products. The method may include amplifying the plurality of extension products using a primer capable of hybridizing to the first universal sequence or its complement and a primer capable of hybridizing to the third universal sequence or its complement, thereby generating a first plurality of barcoded amplicons. Amplifying the plurality of extension products may include adding sequences of binding sites of sequencing primers and / or sequencing adapters, their complementary sequences, and / or portions thereof to the plurality of extension products. The method may include determining the copy number of a nucleic acid target in each of the one or more single cells based on the number of first molecular labels having distinct sequences associated with the first plurality of barcoded amplicons or products thereof. Determining the copy number of a nucleic acid target in each of the one or more single cells may include determining the number of each of a plurality of nucleic acid targets in each of the one or more single cells based on the number of first molecular labels having distinct sequences associated with barcoded amplicons among the first plurality of barcoded amplicons that include the respective sequences of the plurality of nucleic acid targets. The sequence of each of the plurality of nucleic acid targets may include a subsequence of each of the plurality of nucleic acid targets. The sequence of a nucleic acid target in the first plurality of barcoded amplicons may include a subsequence of a nucleic acid target.

[0202] The method may include amplifying a first plurality of barcoded amplicons using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a third universal sequence or its complement, thereby generating a second plurality of barcoded amplicons. Amplifying the first plurality of barcoded amplicons may include adding sequences of sequencing primers and / or sequencing adapter binding sites, their complementary sequences, and / or portions thereof to the first plurality of barcoded amplicons. The method may include determining the copy number of a nucleic acid target in each of one or more single cells based on the number of first molecular labels having distinct sequences associated with the second plurality of barcoded amplicons or products thereof. In some embodiments, the first plurality of barcoded amplicons and / or the second plurality of barcoded amplicons comprise whole transcriptome amplification (WTA) products.

[0203] The method may include synthesizing a third plurality of barcoded amplicons using the plurality of barcoded nucleic acid molecules as templates to generate a third plurality of barcoded amplicons. Synthesizing the third plurality of barcoded amplicons may include performing polymerase chain reaction (PCR) amplification of the plurality of barcoded nucleic acid molecules. Synthesizing the third plurality of barcoded amplicons may include PCR amplification using a primer capable of hybridizing to a first universal sequence or its complement and a target-specific primer. The method may include obtaining sequence information of the third plurality of barcoded amplicons or their products. Obtaining sequence information may include attaching sequencing adapters to the third plurality of barcoded amplicons or their products. The method may include determining the copy number of a nucleic acid target in each of one or more single cells based on the number of first molecular labels having distinct sequences associated with the third plurality of barcoded amplicons or their products.

[0204] The nucleic acid target may include a nucleic acid molecule. The nucleic acid molecule may include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, sample-indexing oligonucleotides, cellular component binding reagent-specific oligonucleotides, or any combination thereof. In some embodiments, extending the plurality of oligonucleotide barcodes includes extending the plurality of oligonucleotide barcodes using a reverse transcriptase and / or a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. The DNA polymerase may include a Klenow fragment. The reverse transcriptase may include a viral reverse transcriptase (e.g., murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase). In some embodiments, the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence are the same. In some embodiments, the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence are different. In some embodiments, the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence comprise a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof. In some embodiments, the sequencing adapter comprises a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof. In some embodiments, the sequencing primer comprises a lead 1 sequencing primer, a lead 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof. In some embodiments, at least 10 of the plurality of oligonucleotide barcodes comprise different first molecular label sequences. In some embodiments, each of the plurality of oligonucleotide barcodes comprises a cell label. Each cell label of the plurality of oligonucleotide barcodes can comprise at least 6 nucleotides. In some embodiments, oligonucleotide barcodes associated with the same third solid support comprise the same cell label.In some embodiments, the oligonucleotide barcodes associated with different third solid supports comprise different cell labels.

[0205] Compositions and Kits In some embodiments, a composition (e.g., a kit) is provided. In some embodiments, the composition comprises a plurality of first solid supports comprising a plurality of capture reagents capable of specifically binding to at least one of a plurality of secreted analytes secreted by a single cell, and a plurality of secreted analyte binding reagents, each capable of specifically binding to the secreted analyte to which the capture reagent is bound, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent. In some embodiments, the secreted analyte binding reagent and the capture reagent are capable of binding to distinct epitopes of the same secreted analyte. The composition may comprise a plurality of second solid supports comprising isolated reagents capable of specifically binding to surface cellular targets.

[0206] The secreted analyte-binding reagent-specific oligonucleotides can include a second molecular beacon sequence. The second molecular beacon sequence can be 2 to 20 nucleotides in length. In some embodiments, the second molecular beacon sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are identical. In some embodiments, the second molecular beacon sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different, and the unique identifier sequences of at least two secreted analyte-binding reagent-specific oligonucleotides are different. The secreted analyte-binding reagent-specific oligonucleotide may comprise a second universal sequence. The second universal sequence may comprise a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof. The sequencing adapter may comprise a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof. The sequencing primer may comprise a lead 1 sequencing primer, a lead 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof.

[0207] The cellular component-binding reagent-specific oligonucleotide may include a poly(dA) region. The secreted analyte-binding reagent-specific oligonucleotide may include an alignment sequence adjacent to the poly(dA) region. The alignment sequence may be one or more nucleotides in length. The alignment sequence may be two or more nucleotides in length. The alignment sequence may include guanine, cytosine, thymine, uracil, or a combination thereof. The alignment sequence may include a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or a combination thereof. The alignment sequence may be 5' to the poly(dA) region.

[0208] The secreted analyte-binding reagent-specific oligonucleotide may be associated with the secreted analyte-binding reagent through a linker. The linker may comprise a carbon chain. The carbon chain may comprise 2 to 30 carbons. The carbon chain may comprise 12 carbons. The linker may comprise a 5' amino modifier C12 (5AmMC12) or a derivative thereof. The secreted analyte-binding reagent-specific oligonucleotide may be attached to the secreted analyte-binding reagent. The secreted analyte-binding reagent-specific oligonucleotide may be covalently bound to the secreted analyte-binding reagent. The secreted analyte-binding reagent-specific oligonucleotide may be non-covalently bound to the secreted analyte-binding reagent. The secreted analyte-binding reagent-specific oligonucleotide may be conjugated to the secreted analyte-binding reagent. The secreted analyte-binding reagent-specific oligonucleotide may be conjugated to the secreted analyte-binding reagent through a chemical group selected from a group cleavable by UV light, streptavidin, biotin, an amine, and combinations thereof.

[0209] The composition may include a DNA polymerase (e.g., Klenow fragment) lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity. The composition may include a reverse transcriptase, such as a viral reverse transcriptase (e.g., murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase). The composition may include a buffer, a cartridge, or both. The composition may include a plurality of oligonucleotide barcodes. The plurality of oligonucleotide barcodes are associated with a third solid support. The composition may include a third solid support. [Example]

[0210] Certain aspects of the above embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way.

[0211] Example 1 Solid support-based compositions and methods This example describes non-limiting exemplary solid support-based compositions and methods provided herein. This example provides data demonstrating the feasibility of loading single calcein-positive peripheral blood mononuclear cells (PBMCs) (green) into a Rhapsody cartridge along with CBA beads (7.5 μm). Figure 6 shows data regarding the use of solid supports provided herein in a Rhapsody cartridge. This example demonstrates that 0.5 μm-sized (Dragon Green) beads can be sequentially included in a Rhapsody cartridge along with cells. Peripheral blood mononuclear cells (PBMCs) were stained with calcein and loaded into a Rhapsody cartridge to obtain single cells. Beads of various sizes and green fluorescence—CBA beads (7.5 μm), 15 μm beads, and Dragon Green beads (0.5 μm)—were sequentially loaded. Images obtained by the Rhapsody scanner were overlaid using ImageJ software. Arrow #1 indicates a calcein-stained cell (bright green). Arrow #2 indicates Dragon Green 0.5 μm beads. Arrow #3 indicates CBA beads (7.5 μm). Arrow #4 indicates 15 μm beads. Furthermore, the data suggest that PBMCs can be cultured in the Rhapsody cartridge for 48 hours.

[0212] Example 2 Proof of principle single-cell secretome workflow The feasibility of the cartridge loading workflow on the BD Rhapsody™ system was tested using the compositions and methods provided herein. The feasibility of the single-cell secretome workflow provided herein was investigated on the BD Rhapsody™ system. Peripheral blood mononuclear cells (PBMCs) bound to magnetic beads (BD iMag) were loaded onto the BD Rhapsody™ cartridge (Figure 7). Single-cell secretome beads (scS beads) were then loaded, followed by BD Rhapsody™ beads (Figure 7). Cell lysis was performed according to the protocol. Finally, complexes of BD Rhapsody™ and scS beads were successfully recovered for downstream library preparation, as evidenced by the BD Rhapsody™ scanner (Figure 7), demonstrating the feasibility of the compositions and methods provided herein.

[0213] Next, we used a novel flow cytometry-based method to validate / QC the single-cell secretome (scS) beads and detection antibodies (Figures 8A-8C). Figure 8A shows a schematic diagram of a non-limiting exemplary workflow. scS beads were generated and incubated with recombinant IFNγ (Figure 8A). The beads were washed to remove unbound IFNγ, and a detection antibody with a unique oligonucleotide sequence was added, followed by the addition of Alexa Fluor 647 (AF647)-dT, which can bind to the detection Ab (Figure 8A). Bead complexes were acquired using flow cytometry to confirm complex formation (Figures 8B-8C). Figure 8B shows that a positive signal was detected upon addition of AF647-dT, suggesting that both the scS beads and the detection Ab are functional. Figure 8C shows that an increase in the AF647 signal was detected with increasing amounts of IFNγ, thereby confirming binding specificity. As seen in Figure 8B, a positive signal was detected, validating the scS beads and detection antibody. As can be seen in Figure 8C, increasing amounts of IFNγ cytokine can be captured by scS beads.

[0214] Finally, the compositions and workflows provided herein were tested using real-time PCR. Figure 9A shows a schematic diagram of a non-limiting exemplary workflow for detecting cytokine secretion using real-time PCR on a BD Rhapsody™ system (Figure 9A). scS beads bound with or without recombinant IFNγ were loaded onto a BD Rhapsody™ cartridge. After addition of a detection antibody, the bead complex was recovered from the cartridge. Real-time PCR was performed using two samples, and a significantly higher level of PCR signal was detected from the "IFN sample" compared to the "no IFN sample." The signal in the "+IFNγ" sample was 2.0 times higher than that in the "-IFNγ" sample. 12 It was double. Collectively, these results provide proof of principle for the single-cell secretome methods and compositions provided herein.

[0215] term In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, except where such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter as defined by the appended claims. Those skilled in the art will recognize that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in differing order. Furthermore, the outlined steps and operations are provided only as examples, and some of the steps and operations may be arbitrarily combined into fewer steps and operations or expanded into additional steps and operations without diminishing the essential elements of the embodiments of the present disclosure.

[0216] In connection with the use of virtually any plural and / or singular term herein, those of skill in the art can convert from plural to singular and / or from singular to plural where appropriate in context and / or application. Various singular / plural permutations may be expressly stated herein for clarity. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference herein to "or" is intended to encompass "and / or" unless specified otherwise.

[0217] In general, it will be understood by those skilled in the art that the terms used herein, particularly in the appended claims (e.g., the body of the appended claims), are generally intended to be “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Furthermore, where a specific number of introduced claim recitations is intended, such intention will be explicitly set forth in the claim; it will be understood by those skilled in the art that, in the absence of such a recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" means that any particular claim containing such introduced claim recitation is limited to embodiments containing only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"), nor should the use of definite articles used to introduce claim recitations.Furthermore, even if a particular number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., an unqualified recitation such as "two recitations" without other modifiers means at least two recitations, or more than two recitations). Furthermore, when a convention similar to "such as at least one of A, B, and C" is used, it is generally intended that such a configuration be understood by one of skill in the art (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). When a convention similar to "such as at least one of A, B, or C" is used, it is generally intended that such construction have the meaning that one of ordinary skill in the art would understand that convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, a system having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). Furthermore, it will be understood by those skilled in the art that virtually any disjunctive word and / or phrase expressing two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0218] Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, one of skill in the art will recognize that the present disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0219] As will be understood by those skilled in the art, for all purposes, e.g., with respect to the provision of a specification, all ranges disclosed herein encompass all possible subranges and combinations of such subranges. Any recited range is readily recognizable as being fully descriptive and that the range can be divided into at least 2, 3, 4, 5, 10, etc. equal parts. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. Similarly, as will be understood by those skilled in the art, terms such as "up to," "at least," "greater than," "less than," etc., all refer to ranges that are inclusive of the recited numbers and that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 items refers to groups having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to groups having 1, 2, 3, 4, or 5 items, and so forth.

[0220] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for measuring the copy number of a secreted analyte secreted by a single cell, comprising: distributing the plurality of first solid supports and the one or more single cells into a plurality of compartments, wherein a compartment of the plurality of compartments comprises one or more first solid supports of the plurality of first solid supports and a single cell of the one or more single cells, wherein the one or more single cells are capable of secreting a plurality of secreted analytes, and each of the first solid supports comprises a plurality of capture reagents capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell; contacting one or more first solid supports with a plurality of secreted analyte binding reagents, each of the plurality of secreted analyte binding reagents capable of specifically binding to the secreted analyte bound by the capture reagent, and each of the plurality of secreted analyte binding reagents comprising a secreted analyte binding reagent-specific oligonucleotide comprising a unique analyte identifier sequence for the secreted analyte binding reagent; contacting a plurality of oligonucleotide barcodes with secreted analyte-binding reagent-specific oligonucleotides for hybridization, each of the oligonucleotide barcodes comprising a first molecular label; extending the plurality of oligonucleotide barcodes hybridized to the secreted analyte-binding reagent-specific oligonucleotides to generate a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each barcoded secreted analyte-binding reagent-specific oligonucleotides comprising a sequence complementary to at least a portion of the unique analyte identifier sequence and a first molecular label; obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one secreted analyte secreted by each of the one or more single cells; A method comprising:

2. 1. A method for determining the copy number of a secreted analyte secreted by a single cell and the copy number of a nucleic acid target in a single cell, comprising: distributing the plurality of first solid supports and the one or more single cells into a plurality of compartments, wherein a compartment of the plurality of compartments comprises one or more first solid supports of the plurality of first solid supports and a single cell of the one or more single cells, the one or more single cells comprising a copy of a nucleic acid target, the one or more single cells being capable of secreting a plurality of secreted analytes, and each of the first solid supports comprising a plurality of capture reagents capable of specifically binding to at least one of the plurality of secreted analytes secreted by the single cell; contacting one or more first solid supports with a plurality of secreted analyte binding reagents, each capable of specifically binding to a capture reagent-bound secreted analyte, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide that comprises a unique analyte identifier sequence for the secreted analyte binding reagent; contacting a plurality of oligonucleotide barcodes for hybridization with copies of a secreted analyte-binding reagent-specific oligonucleotide and a nucleic acid target, wherein each oligonucleotide barcode comprises a first molecular label; extending the plurality of oligonucleotide barcodes hybridized to copies of the nucleic acid target to generate a plurality of barcoded nucleic acid molecules, each of which comprises a sequence complementary to at least a portion of the nucleic acid target and a first molecular label; extending the plurality of oligonucleotide barcodes hybridized to the secreted analyte-binding reagent-specific oligonucleotides to generate a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides, each barcoded secreted analyte-binding reagent-specific oligonucleotides comprising a sequence complementary to at least a portion of the unique analyte identifier sequence and a first molecular label; obtaining sequence information of a plurality of barcoded nucleic acid molecules or products thereof to determine the copy number of the nucleic acid target in each of the one or more single cells; obtaining sequence information of the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one secreted analyte secreted by each of the one or more single cells; A method comprising:

3. The one or more single cells include one or more single cells associated with a second solid support, and the method further comprises, prior to the distributing step: contacting the population of single cells with a plurality of second solid supports to generate one or more single cells associated with the second solid supports, wherein the one or more single cells comprise a surface cell target, and each of the second solid supports comprises a plurality of isolation reagents, each of the plurality of isolation reagents being capable of specifically binding to the surface cell target; The method may include removing single cells of the population of single cells that are not associated with the second solid support; The method according to any one of claims 1 to 2.

4. 4. The method of any one of claims 1 to 3, wherein the one or more single cells are one or more cell types of interest, and the cell types of interest optionally comprise surface cellular targets to which the isolation reagent of the second solid support can bind.

5. 5. The method of claim 1, further comprising, after the step of contacting the one or more first solid supports with the plurality of secreted analyte binding reagents, removing one or more secreted analyte binding reagents of the plurality of secreted analyte binding reagents that have not been contacted with the one or more first solid supports.

6. 6. The method of any one of claims 1 to 5, wherein removing one or more secreted analyte binding reagents that are not in contact with one or more first solid supports comprises removing one or more secreted analyte binding reagents that are not in contact with at least one of the respective capture reagent-bound secreted analytes.

7. The method of any one of claims 1 to 6, wherein the first solid support and / or the second solid support comprises a magnetic material, optionally a ferromagnetic material.

8. 8. The method of any one of claims 1 to 7, wherein the removing step comprises applying a magnetic field to the plurality of compartments, and wherein the single cell associated with the second solid support and the one or more first solid supports may be able to remain in the compartment when the magnetic field is applied.

9. 9. The method of any one of claims 1 to 8, comprising one or more incubation steps for a period of time, which may be about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, about 120 minutes, or about 240 minutes, and further wherein the incubation may occur after the step of (i) contacting the single cell with the first solid support and / or after the step of (ii) contacting the one or more first solid supports with the plurality of secreted analyte-binding reagents.

10. 10. The method of any one of claims 1 to 9, wherein a compartment of the plurality of compartments comprises from about 2 to about 20 first solid supports, optionally about 8 first solid supports, which first solid supports may be the same or different, and further wherein each first solid support may comprise a single type of capture reagent and / or may comprise different types of capture reagents.

11. The method of any one of claims 1 to 10, wherein the first solid support and / or the second solid support is less than about 15 μm.

12. 12. The method of any one of claims 1 to 11, comprising lysing a single cell in the compartment, wherein lysing the single cell may comprise heating the sample, contacting the sample with a detergent, altering the pH of the sample, or any combination thereof.

13. 13. The method of any one of claims 1 to 12, wherein the one or more single cells comprise T cells, B cells, tumor cells, bone marrow cells, blood cells, normal cells, fetal cells, maternal cells, or mixtures thereof.

14. 14. The method of any one of claims 1 to 13, wherein the at least one secreted analyte comprises (a) a lymphokine, an interleukin, a chemokine, or any combination thereof, and / or (b) a cytokine, a hormone, a molecular toxin, or any combination thereof.

15. 15. The method of any one of claims 1 to 14, wherein the at least one secreted analyte comprises nerve growth factor, liver growth factor, fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor, osteoinductive factor, interferon, colony-stimulating factor, or any combination thereof.

16. The method of any one of claims 1 to 15, wherein the secreted analyte binding reagent and the capture reagent are capable of binding to distinct epitopes of the same secreted analyte.

17. 17. The method of any one of claims 1 to 16, wherein one or more of the secreted analyte binding reagent, capture reagent, and isolation reagent comprise an antibody or fragment thereof, and the antibody or fragment thereof may comprise a monoclonal antibody.

18. 18. The method of any one of claims 1 to 17, wherein the capture reagent and / or isolation reagent are conjugated to the first solid support and / or the second solid support by a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilic substitution reaction, a non-aldol carbonyl reaction, a carbon-carbon multiple bond addition, an oxidation reaction, a click reaction, or any combination thereof.

19. 19. The method of any one of claims 1 to 18, wherein the surface cellular target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

20. 20. The method of any one of claims 1-19, wherein a plurality of oligonucleotide barcodes are associated with a third solid support, and a compartment of the plurality of compartments comprises a single third solid support.

21. The method according to any one of claims 1 to 20, wherein the compartment is a well or a droplet.

22. 22. The method of any one of claims 1 to 21, wherein each oligonucleotide barcode comprises a first universal sequence.

23. 23. The method of any one of claims 1 to 22, wherein the oligonucleotide barcode comprises a target binding region comprising a capture sequence, the target binding region may comprise a poly(dT) region, the secreted analyte-binding reagent-specific oligonucleotide may further comprise a sequence complementary to the capture sequence configured to capture the secreted analyte-binding reagent-specific oligonucleotide, and the sequence complementary to the capture sequence may comprise a poly(dA) region.

24. 24. The method of any one of claims 1 to 23, wherein the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides comprises the complement of a first universal sequence.

25. The method of any one of claims 1 to 24, wherein the secreted analyte-binding reagent-specific oligonucleotide comprises a second universal sequence.

26. obtaining sequence information of a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof, amplifying the plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a second universal sequence or its complement to generate a plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides; obtaining sequencing data for the plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof; The method of any one of claims 1 to 25, comprising:

27. The method of any one of claims 1 to 26, wherein the secreted analyte-binding reagent-specific oligonucleotide comprises a second molecular beacon.

28. 28. The method of any one of claims 1 to 27, wherein at least 10 of the plurality of secreted analyte-binding reagent-specific oligonucleotides comprise different second molecular beacon sequences.

29. 30. The method of claim 28, wherein the second molecular beacon sequences of at least two secreted analyte binding reagent-specific oligonucleotides are different and the unique analyte identifier sequences of at least two secreted analyte binding reagent-specific oligonucleotides are identical.

30. 30. The method of claim 28, wherein the second molecular beacon sequences of at least two secreted analyte binding reagent-specific oligonucleotides are different and the unique analyte identifier sequences of at least two secreted analyte binding reagent-specific oligonucleotides are different.

31. 31. The method of any one of claims 1 to 30, wherein the number of unique first molecular label sequences associated with unique analyte identifier sequences for secreted analyte binding reagents capable of specifically binding to the at least one secreted analyte in the sequencing data indicates the copy number of the at least one secreted analyte secreted by each of the one or more single cells.

32. 32. The method of any one of claims 27 to 31, wherein the number of unique second molecular label sequences associated with unique analyte identifier sequences for secreted analyte binding reagents capable of specifically binding to the at least one secreted analyte in the sequencing data indicates the copy number of the at least one secreted analyte secreted by each of the one or more single cells.

33. 33. The method of any one of claims 1 to 32, comprising determining the copy number of at least one secreted analyte secreted by each of the one or more single cells based on the number of first molecular labels and / or second molecular labels having distinct sequences associated with a plurality of barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof.

34. 34. The method of any one of claims 26 to 33, comprising determining the copy number of at least one secreted analyte secreted by each of the one or more single cells based on the number of first molecular labels and / or second molecular labels having distinct sequences associated with a plurality of amplified barcoded secreted analyte-binding reagent-specific oligonucleotides or products thereof.

35. 35. The method of any one of claims 1 to 34, wherein obtaining sequence information comprises attaching sequencing adaptors to a plurality of barcoded secreted analyte binding reagent-specific oligonucleotides or products thereof.

36. the secreted analyte binding reagent-specific oligonucleotide comprises an alignment sequence flanking the poly(dA) tract, the alignment sequence may be one or more nucleotides in length, or two or more nucleotides in length; and (a) the alignment sequence may contain guanine, cytosine, thymine, uracil, or a combination thereof; (b) the alignment sequence may include a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or a combination thereof; and / or (c) the alignment sequence may be 5' to the poly(dA) region; 36. The method according to any one of claims 1 to 35.

37. 37. The method of any one of claims 1 to 36, wherein the secreted analyte binding reagent-specific oligonucleotide is associated with the secreted analyte binding reagent through a linker, wherein the linker may comprise a carbon chain or a 5' amino modifier C12 (5AmMC12) or a derivative thereof, wherein the carbon chain may comprise from 2 to 30 carbons, and further wherein the carbon chain may comprise 12 carbons.

38. A method according to any preceding claim, wherein the secreted analyte binding reagent-specific oligonucleotide is configured to be detachable from the secreted analyte binding reagent.

39. A method according to any preceding claim, comprising the step of dissociating the secreted analyte-binding reagent-specific oligonucleotide from the secreted analyte-binding reagent.

40. 40. The method of any one of claims 1 to 39, comprising isolating a complex from one or more of the plurality of compartments, wherein the complex may comprise the third solid support, the first solid support, and / or the secreted analyte-binding reagent.

41. 41. The method of any one of claims 2 to 40, wherein determining the copy number of the nucleic acid target in each of the one or more single cells comprises determining the copy number of the nucleic acid target in each of the one or more single cells based on the number of first molecular labels having distinct sequences, their complements, or combinations thereof, associated with a plurality of barcoded nucleic acid molecules or products thereof.

42. contacting random primers with a plurality of barcoded nucleic acid molecules, wherein each of the random primers comprises a third universal sequence or a complement thereof; extending random primers hybridized to the plurality of barcoded nucleic acid molecules to generate a plurality of extension products; The method of any one of claims 2 to 41, comprising:

43. 43. The method of claim 42, comprising amplifying a plurality of extension products using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a third universal sequence or its complement, thereby generating a first plurality of barcoded amplicons, wherein amplifying the plurality of extension products may comprise adding sequences of sequencing primer and / or sequencing adapter binding sites, their complements, and / or portions thereof to the plurality of extension products.

44. 44. The method of Claim 43, comprising determining the copy number of the nucleic acid target in each of the one or more single cells based on the number of first molecular labels having distinct sequences associated with the first plurality of barcoded amplicons or products thereof.

45. 45. The method of any one of claims 43-44, wherein determining the copy number of the nucleic acid target in each of the one or more single cells comprises determining the number of each of the plurality of nucleic acid targets in each of the one or more single cells based on a number of first molecular labels having distinct sequences associated with barcoded amplicons of a first plurality of barcoded amplicons comprising the sequences of each of the plurality of nucleic acid targets, wherein the sequences of each of the plurality of nucleic acid targets may comprise subsequences of each of the plurality of nucleic acid targets.

46. 46. ​​The method of any one of claims 43 to 45, wherein the sequence of the nucleic acid target in the first plurality of barcoded amplicons comprises a subsequence of the nucleic acid target.

47. 47. The method of any one of claims 43 to 46, comprising amplifying a first plurality of barcoded amplicons using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a third universal sequence or its complement, thereby generating a second plurality of barcoded amplicons, wherein amplifying the first plurality of barcoded amplicons optionally comprises adding sequencing primer and / or sequencing adapter binding site sequences, complementary sequences thereof, and / or portions thereof to the first plurality of barcoded amplicons.

48. 48. The method of Claim 47, comprising determining the copy number of the nucleic acid target in each of the one or more single cells based on the number of first molecular labels having distinct sequences associated with the second plurality of barcoded amplicons or products thereof.

49. 49. The method of any one of claims 43-48, wherein the first plurality of barcoded amplicons and / or the second plurality of barcoded amplicons comprise whole transcriptome amplification (WT A) products.

50. 50. The method of any one of claims 1 to 49, comprising synthesizing a third plurality of barcoded amplicons using the plurality of barcoded nucleic acid molecules as templates to generate a third plurality of barcoded amplicons, wherein synthesizing the third plurality of barcoded amplicons may comprise performing polymerase chain reaction (PCR) amplification of the plurality of barcoded nucleic acid molecules, and wherein synthesizing the third plurality of barcoded amplicons may comprise PCR amplification using a primer capable of hybridizing to the first universal sequence or its complement and a target-specific primer.

51. 51. The method of claim 50, comprising obtaining sequence information of the third plurality of barcoded amplicons or products thereof, wherein obtaining sequence information may comprise attaching sequencing adaptors to the third plurality of barcoded amplicons or products thereof.

52. 52. The method of any one of claims 50-51, comprising determining the copy number of the nucleic acid target in each of the one or more single cells based on the number of first molecular labels having distinct sequences associated with the third plurality of barcoded amplicons or products thereof.

53. 53. The method of any one of claims 2 to 52, wherein the nucleic acid target comprises a nucleic acid molecule, which may comprise ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA comprising a poly(A) tail, a sample-indexing oligonucleotide, a cellular component-binding reagent-specific oligonucleotide, or any combination thereof.

54. one or more single cells contain multiple cellular component targets; contacting a plurality of cellular component binding reagents with one or more single cells, each of the plurality of cellular component binding reagents comprising a cellular component binding reagent-specific oligonucleotide comprising a unique identifier sequence for the cellular component binding reagent, the cellular component binding reagent capable of specifically binding to at least one of a plurality of cellular component targets; contacting a plurality of oligonucleotide barcodes with cellular component binding reagent-specific oligonucleotides for hybridization, each of the oligonucleotide barcodes comprising a molecular label and a first universal sequence; extending the plurality of oligonucleotide barcodes hybridized to the cellular component binding reagent-specific oligonucleotides to generate a plurality of barcoded cellular component binding reagent-specific oligonucleotides, each of which comprises a sequence complementary to at least a portion of the unique identifier sequence and a molecular label; obtaining sequence information of the plurality of barcoded cellular component binding reagent-specific oligonucleotides or products thereof to determine the copy number of at least one cellular component target among the plurality of cellular component targets in each of the one or more single cells; 54. The method of any one of claims 1 to 53, further comprising:

55. 55. The method of claim 54, wherein the cellular component binding reagent-specific oligonucleotide comprises a sequence complementary to a capture sequence configured to capture the cellular component binding reagent-specific oligonucleotide.

56. The method of any one of claims 54 to 55, wherein the cell component binding reagent-specific oligonucleotide comprises a fourth universal sequence.

57. obtaining sequence information of a plurality of barcoded cellular component binding reagent-specific oligonucleotides or products thereof, amplifying the plurality of barcoded cellular component binding reagent-specific oligonucleotides or products thereof using a primer capable of hybridizing to the first universal sequence or its complement and a primer capable of hybridizing to the fourth universal sequence or its complement to generate a plurality of amplified barcoded cellular component binding reagent-specific oligonucleotides; obtaining sequencing data for the plurality of amplified barcoded cellular component binding reagent-specific oligonucleotides or products thereof; 57. The method of any one of claims 54 to 56, comprising:

58. 58. The method of any one of claims 54 to 57, wherein obtaining sequence information comprises attaching sequencing adaptors to a plurality of barcoded cellular component binding reagent-specific oligonucleotides or products thereof.

59. The method according to any one of claims 54 to 58, further comprising, after the step of contacting the plurality of cellular component binding reagents with one or more single cells, a step of removing one or more cellular component binding reagents from the plurality of cellular component binding reagents that have not come into contact with one or more single cells, wherein the step of removing one or more cellular component binding reagents that have not come into contact with one or more single cells may comprise a step of removing one or more cellular component binding reagents that have not come into contact with at least one of the plurality of cellular component targets, respectively.

60. 60. The method of any one of claims 54 to 59, wherein the cellular component target comprises an intracellular protein, a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

61. 61. The method of any one of claims 54 to 60, wherein the cellular component target comprises a housekeeping protein, detection of which indicates the presence of a single cell in the compartment.

62. 62. The method of any one of claims 1 to 61, wherein extending the plurality of oligonucleotide barcodes comprises extending the plurality of oligonucleotide barcodes using a reverse transcriptase and / or a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity, wherein the DNA polymerase may comprise Klenow fragment; and / or the reverse transcriptase comprises a viral reverse transcriptase, wherein the viral reverse transcriptase may be murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase.

63. 63. The method of any one of claims 1 to 62, wherein the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence are the same.

64. 63. The method of any one of claims 1 to 62, wherein the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence are different.

65. 65. The method of any one of claims 1 to 64, wherein the first universal sequence, the second universal sequence, the third universal sequence, and / or the fourth universal sequence comprise a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof, wherein the sequencing adapter may comprise a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof, and further wherein the sequencing primer may comprise a lead 1 sequencing primer, a lead 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof.

66. 66. The method of any one of claims 1-65, wherein at least 10 of the plurality of oligonucleotide barcodes comprise different first molecular label sequences.

67. 67. The method of any one of claims 1-66, wherein the plurality of oligonucleotide barcodes each comprise a cell label, and wherein each cell label of the plurality of oligonucleotide barcodes may comprise at least 6 nucleotides, and wherein oligonucleotide barcodes associated with the same third solid support may comprise the same cell label, and further wherein oligonucleotide barcodes associated with different third solid supports may comprise different cell labels.

68. 68. The method of any one of claims 1 to 67, wherein the first solid support, the second solid support, and / or the third solid support comprise a synthetic particle or a planar surface, and wherein at least one of the plurality of oligonucleotide barcodes may be immobilized or partially immobilized on a synthetic particle, or wherein at least one of the plurality of oligonucleotide barcodes may be encapsulated or partially encapsulated within a synthetic particle, and wherein the synthetic particle may be disintegrable.

69. The composite particles include beads, the beads being Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; or Collapsible hydrogel particles 69. The method of claim 68, which may include:

70. each of the plurality of oligonucleotide barcodes comprises a linker functional group; the synthetic particles comprise solid support functional groups; the support functional group and the linker functional group are associated with each other; The linker functional group and the support functional group may each be selected from the group consisting of C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof; 70. The method of any one of claims 68 to 69.

71. each of the plurality of isolating reagents comprises a linker functional group; the synthetic particles comprise solid support functional groups; the support functional group and the linker functional group are associated with each other; The linker functional group and the support functional group may each be selected from the group consisting of C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof; 70. The method of any one of claims 68 to 69.

72. each of the plurality of capture reagents comprises a linker functional group; the synthetic particles comprise solid support functional groups; the support functional group and the linker functional group are associated with each other; The linker functional group and the support functional group may each be selected from the group consisting of C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof; 70. The method of any one of claims 68 to 69.

73. a plurality of first solid supports comprising a plurality of capture reagents capable of specifically binding to at least one of a plurality of secreted analytes secreted by a single cell; a plurality of secreted analyte binding reagents, each capable of specifically binding to the capture reagent-bound secreted analyte, wherein each of the plurality of secreted analyte binding reagents comprises a secreted analyte binding reagent-specific oligonucleotide that includes a unique analyte identifier sequence for the secreted analyte binding reagent; A composition comprising: A composition wherein the secreted analyte binding reagent and the capture reagent are capable of binding to distinct epitopes of the same secreted analyte.

74. 74. The composition of claim 73, wherein the secreted analyte-binding reagent-specific oligonucleotide comprises a second molecular beacon sequence, and the second molecular beacon sequence may be from 2 to 20 nucleotides in length.

75. 75. The composition of claim 74, wherein the second molecular beacon sequences of at least two secreted analyte binding reagent-specific oligonucleotides are different and the unique analyte identifier sequences of at least two secreted analyte binding reagent-specific oligonucleotides are identical.

76. 75. The composition of claim 74, wherein the second molecular beacon sequences of at least two secreted analyte binding reagent-specific oligonucleotides are different and the unique analyte identifier sequences of at least two secreted analyte binding reagent-specific oligonucleotides are different.

77. 77. The composition of any one of claims 73 to 76, wherein the secreted analyte-binding reagent-specific oligonucleotide comprises a second universal sequence, and the second universal sequence may comprise a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof, and further wherein (1) the sequencing adapter may comprise a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof; or (2) the sequencing primer may comprise a lead 1 sequencing primer, a lead 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof.

78. 78. The composition of any one of claims 73 to 77, wherein the secreted analyte binding reagent-specific oligonucleotide comprises a poly(dA) region.

79. 79. The composition of any one of claims 73 to 78, wherein the secreted analyte binding reagent-specific oligonucleotide comprises an alignment sequence flanking the poly(dA) tract, and the alignment sequence may be one or more nucleotides in length, and further, the alignment sequence may be two or more nucleotides in length.

80. (a) the alignment sequence contains guanine, cytosine, thymine, uracil, or a combination thereof; (b) the alignment sequence comprises a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, a poly(dU) sequence, or a combination thereof; and / or (c) the alignment sequence is 5' to the poly(dA) region; 80. The composition of claim 79.

81. 81. The composition of any one of claims 73 to 80, wherein the secreted analyte binding reagent-specific oligonucleotide is associated with the secreted analyte binding reagent through a linker, and the linker may comprise a carbon chain or a 5' amino modifier C12 (5AmMC12) or a derivative thereof, and the carbon chain may comprise from 2 to 30 carbons, and further, the carbon chain may comprise 12 carbons.

82. 82. The composition of any one of claims 73 to 81, wherein the secreted analyte-binding reagent-specific oligonucleotide is bound to the secreted analyte-binding reagent, and the secreted analyte-binding reagent-specific oligonucleotide may be (1) covalently bound to the secreted analyte-binding reagent, or (2) non-covalently bound to the secreted analyte-binding reagent.

83. 83. The composition of any one of claims 73 to 82, wherein the secreted analyte binding reagent-specific oligonucleotide is conjugated to the secreted analyte binding reagent, and optionally the secreted analyte binding reagent-specific oligonucleotide is conjugated to the secreted analyte binding reagent through a chemical group selected from the group consisting of a group cleavable by UV light, streptavidin, biotin, an amine, and combinations thereof.

84. 84. The composition of any one of claims 73-83, wherein the secreted analyte comprises (a) a lymphokine, an interleukin, a chemokine, or any combination thereof, or (b) a cytokine, a hormone, a molecular toxin, or any combination thereof.

85. 85. The composition of any one of claims 73-84, wherein the secreted analyte comprises nerve growth factor, liver growth factor, fibroblast growth factor, vascular endothelial growth factor, platelet-derived growth factor, transforming growth factor, osteoinductive factor, interferon, colony-stimulating factor, or any combination thereof.

86. 86. The composition of any one of claims 73 to 85, comprising a second solid support comprising an isolation reagent capable of specifically binding to a surface cell target.

87. 87. The composition of any one of claims 73 to 86, wherein the capture reagent and / or isolation reagent is conjugated to the first solid support and / or the second solid support by a 1,3-dipolar cycloaddition reaction, a hetero-Diels-Alder reaction, a nucleophilic substitution reaction, a non-aldol carbonyl reaction, a carbon-carbon multiple bond addition, an oxidation reaction, a click reaction, or any combination thereof.

88. 88. The composition of any one of claims 73-87, wherein the surface cellular target comprises a carbohydrate, a lipid, a protein, an extracellular protein, a cell surface protein, a cell marker, a B cell receptor, a T cell receptor, a major histocompatibility complex, a tumor antigen, a receptor, an intracellular protein, or any combination thereof.

89. 89. The composition of any one of claims 73 to 88, comprising: (a) a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity, wherein the DNA polymerase optionally comprises a Klenow fragment; and / or (b) a reverse transcriptase, wherein the reverse transcriptase optionally comprises a viral reverse transcriptase, wherein the viral reverse transcriptase optionally comprises murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase.

90. 90. The composition of any one of claims 73-89, comprising: (a) a buffer, a cartridge, or both; and / or (b) a plurality of oligonucleotide barcodes, each oligonucleotide barcode of the plurality of oligonucleotide barcodes comprising a target binding region, wherein the target binding region may comprise a poly(dA) region, a poly(dT) region, a random sequence, a gene-specific sequence, or any combination thereof.

91. 91. The composition of claim 90, wherein the plurality of oligonucleotide barcodes each comprise a molecular label, the molecular label optionally comprising at least six nucleotides, and at least ten of the plurality of oligonucleotide barcodes optionally comprising different molecular label sequences.

92. 92. The composition of any one of claims 90-91, wherein the plurality of oligonucleotide barcodes is associated with a third solid support.

93. 93. The composition of any one of claims 90-92, wherein the plurality of oligonucleotide barcodes each comprise a cellular label, and wherein oligonucleotide barcodes among the plurality of oligonucleotide barcodes associated with the same third solid support may comprise the same cellular label, and wherein oligonucleotide barcodes among the plurality of oligonucleotide barcodes associated with different third solid supports may comprise different cellular labels.

94. 94. The composition of any one of claims 73 to 93, wherein the first solid support, the second solid support, and / or the third solid support comprise a synthetic particle or a planar surface, and wherein at least one of the plurality of oligonucleotide barcodes may be immobilized or partially immobilized on a synthetic particle, or wherein at least one of the plurality of oligonucleotide barcodes may be encapsulated or partially encapsulated within a synthetic particle, and wherein the synthetic particle may be disintegrable.

95. The composite particles include beads, the beads being Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A conjugated beads, protein G conjugated beads, protein A / G conjugated beads, protein L conjugated beads, oligo(dT) conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; or Collapsible hydrogel particles 95. The composition of claim 94, which may comprise:

96. each of the plurality of oligonucleotide barcodes comprises a linker functional group; the synthetic particles comprise solid support functional groups; the support functional group and the linker functional group are associated with each other; The linker functional group and the support functional group may each be selected from the group consisting of C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof; The composition according to any one of claims 94 to 95.

97. each of the plurality of isolating reagents comprises a linker functional group; the synthetic particles comprise solid support functional groups; the support functional group and the linker functional group are associated with each other; The linker functional group and the support functional group may each be selected from the group consisting of C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof; The composition according to any one of claims 94 to 96.

98. each of the plurality of capture reagents comprises a linker functional group; the synthetic particles comprise solid support functional groups; the support functional group and the linker functional group are associated with each other; The linker functional group and the support functional group may each be selected from the group consisting of C6, biotin, streptavidin, a primary amine, an aldehyde, a ketone, and any combination thereof; The composition according to any one of claims 94 to 97.

99. 99. The composition of any one of claims 73-98, wherein the first solid support and / or the second solid support (a) comprises a magnetic material, optionally a ferromagnetic material; and / or (b) is less than about 15 μm.

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