Nuclear barcoding and capture in single cells

Abstract

To provide a method for determining the numbers of targets in a plurality of cells.SOLUTION: The method comprises: isolating a plurality of nuclei of a plurality of cells using a nuclei-isolation composition, where the nuclei-isolation composition comprises a nuclei-binding reagent, and where the nuclei-binding reagent is capable of specifically binding to one or more components of a nucleus; barcoding a plurality of targets in the nuclei using a plurality of barcodes to generate a plurality of barcoded targets, where each of the barcodes comprises a molecular label sequence and a target-binding region, and where the molecular label sequences of at least two of the barcodes comprise different sequences; obtaining sequencing data of the barcoded targets; and estimating the number of each of the targets in the cells using the molecular label sequences of the barcodes in the sequencing data.SELECTED DRAWING: Figure 4A

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. 62 / 714,222, filed August 3, 2018, 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 barcoding of targets, and more specifically to nuclear barcoding. [Background technology]

[0002] Methods and techniques such as barcoding using beads linked to nucleic acid barcodes are useful for single-cell analysis, for example, using reverse transcription, polymerase chain reaction (PCR) amplification, and next-generation sequencing (NGS) to analyze gene expression profiles to determine the state of single cells. However, preparing single-cell suspensions for single-cell analysis can be difficult. Summary of the Invention

[0003] The present disclosure includes embodiments of a method for determining the number of targets in a plurality of cells.In some embodiments, the method includes: using a nucleus isolation composition to isolate a plurality of nuclei from a plurality of cells, the nucleus isolation composition comprises a nucleus binding reagent, and the nucleus binding reagent can specifically bind to one or more components of the nuclei; using a plurality of barcodes to barcode the plurality of targets in the plurality of nuclei to generate a plurality of barcoded targets, each of the plurality of barcodes comprises a molecular beacon sequence and a target binding region, and the molecular beacon sequences of at least two barcodes among the plurality of barcodes comprise different sequences; obtaining sequencing data of the plurality of barcoded targets; and using the molecular beacon sequences of the plurality of barcodes in the sequencing data to estimate the number of each of the plurality of targets in the plurality of cells. In some embodiments, isolating the plurality of nuclei comprises contacting a plurality of nuclei from a plurality of cells with a nuclei isolation composition to generate nuclei bound to a nuclei-binding reagent. Isolating the plurality of nuclei may comprise isolating the nuclei bound to the nuclei-binding reagent using a reagent capable of specifically binding to the nuclei-binding reagent. In some embodiments, the nucleus-binding reagent is associated with a first epitope, and the reagent capable of specifically binding to the nucleus-binding reagent comprises a first epitope-binding reagent. The first epitope may comprise biotin, a hapten, or a combination thereof. The hapten may comprise digoxigenin, 2,4-dinitrophenol, fluorescein, or a combination thereof. The reagent capable of specifically binding to the nucleus-binding reagent may comprise an anti-hapten antibody. The reagent capable of specifically binding to the nucleus-binding reagent may comprise avidin, streptavidin, neutravidin, or a combination thereof. In some embodiments, the nucleus-binding reagent comprises a functional group selected from the group consisting of biotin, streptavidin, heparin, an aptamer, a click chemistry moiety, digoxigenin, a primary amine, a carboxyl, a hydroxyl, an aldehyde, a ketone, or a combination thereof. In some embodiments, the reagent capable of specifically binding to the core-binding reagent comprises a functional group selected from the group consisting of biotin, streptavidin, heparin, an aptamer, a click chemistry moiety, digoxigenin, a primary amine, a carboxyl, a hydroxyl, an aldehyde, a ketone, or a combination thereof.

[0004] In some embodiments, the nucleus-binding reagent comprises a primary antibody capable of specifically binding to one or more components of the nucleus, and the reagent capable of specifically binding to the nucleus-binding reagent comprises a secondary antibody capable of specifically binding to the primary antibody.

[0005] In some embodiments, the nucleic-binding reagent comprises a carbohydrate-binding reagent. The carbohydrate-binding reagent may comprise a carbohydrate-binding protein. The carbohydrate-binding protein may comprise a lectin. The lectin may comprise a mannose-binding lectin, a galactose-binding lectin, an N-acetylgalactosamine-binding lectin, an N-acetylglucosamine-binding lectin, an N-acetylneuraminic acid-binding lectin, a fucose-binding lectin, or a combination thereof. The lectin may include concanavalin A (ConA), lentil lectin (LCH), snowdrop lectin (GNA), castor bean (Ricinus communis) agglutinin (RCA), peanut agglutinin (PNA), jacalin (AIL), hairy vetch lectin (VVL), wheat germ agglutinin (WGA), elderberry lectin (SNA), Maackia amurensis leukoagglutinin (MAL), Maackia amurensis erythrocyte agglutinin (MAH), Ulex europaeus agglutinin (UEA), Aleuria aurantia lectin (AAL), or a combination thereof. The lectin may be or include an agglutinin. The agglutinin may be or include wheat germ agglutinin (WGA). The carbohydrate-binding protein may be derived from or derived from an animal, a bacterium, a virus, a fungus, or a combination thereof. The carbohydrate-binding protein may be derived from or derived from a plant. The plant may be jack bean (Canavalia ensiformis), lentil (Lens culinaris), snowdrop (Galanthus nivalis), castor bean (Ricinus communis), peanut (Arachis hypogaea), jackfruit (Artocarpus integrifolia), vicia villosa, wheat (Triticum vulgaris), elderberry (Sambucus nigra), maackia amurensis, gorse (Ulex europaeus), aleuria aurantia, or a combination thereof.

[0006] In some embodiments, one or more components of the core include a sugar, an oligosaccharide, a polysaccharide, a derivative thereof, or a combination thereof. One or more components of the core may include a monosaccharide, a disaccharide, a polyol, a maltooligosaccharide, a non-maltooligosaccharide, a starch, a non-starch polysaccharide, a derivative thereof, or a combination thereof. One or more components of the core may include glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, maltodextrin, raffinose, stachyose, fructooligosaccharide, amylose, amylopectin, modified starch, glycogen, cellulose, hemicellulose, pectin, a hydrocolloid, a derivative thereof, or a combination thereof. One or more components of the core may be α-D-mannosyl residues, α-D-glucosyl residues, branched α-mannosidic structures of high α-mannose types, branched α-mannosidic structures of hybrid and biantennary complex N-glycans, fucosylated core regions of biantennary and triantennary complex N-glycans, α1-3 and α1-6 linked high mannose structures, Galβ1-4GalNAcβ1-R, Galβ1-3GalNAcα1-Ser / Thr, (Sia)Galβ1-3GalNAcα1-Ser / Thr, GalNAcα-Ser / Thr, GlcNAcβ1-4GlcNAcβ1-4GlcNAc, Neu5Ac (sialic acid), Neu5Acα2-6Gal(NAc)-R, Neu5Ac / Gcα2,3Galβ1,4Glc(NAc), Neu5Ac / Gcα2,3Galβ1,3(Neu5Acα2,6)GalNac, Fucα1-2Gal-R, Fucα1-2Galβ1-4(Fucα1-3 / 4)Galβ1-4GlcNAc, R2-GlcNAcβ1-4(Fucα1-6)GlcNAc-R1, derivatives thereof, or combinations thereof. One or more components of the core may include a glycoprotein, a glycolipid, or a combination thereof.

[0007] In some embodiments, the one or more components of the nucleus include lamin, emerin, nesprin, nurim, UNC-83, Klar, ZYG-12, Kms1p, UNC-84, Klaroid, SUN-1, Sad1p, LBR, MAN1, LAP1, LAP2, LINK, the nuclear pore complex, a portion thereof, or a combination thereof.

[0008] In some embodiments, the nucleus-binding reagent is associated with the nucleus isolation particle. The reagent capable of specifically binding to the nucleus-binding reagent may be associated with the nucleus isolation particle. The reagent capable of specifically binding to the nucleus-binding reagent may be immobilized or partially immobilized to the nucleus isolation particle. For example, the reagent capable of specifically binding to the nucleus-binding reagent may be reversibly, irreversibly, covalently, non-covalently associated with the nucleus isolation particle, or a combination thereof. As another example, the reagent capable of specifically binding to the nucleus-binding reagent may be embedded, partially embedded, not embedded, encapsulated, partially encapsulated, not encapsulated, or a combination thereof in the nucleus isolation particle. In some embodiments, the reagent capable of specifically binding to the nucleus-binding reagent is associated with the nucleus isolation particle through a cleavable linker. The cleavable linker may include, for example, a chemically cleavable linkage, a photocleavable linkage, an acid-labile linker, a heat-sensitive linkage, an enzymatically cleavable linkage, or a combination thereof.

[0009] In some embodiments, the nucleus isolation particles comprise nucleus isolation beads. The nucleus isolation particles 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. The nucleus isolation particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The nucleus isolation particles may be disintegrable. The nucleus isolation particles may comprise disintegrable nucleus isolation hydrogel particles. In some embodiments, isolating nuclei bound to the nuclei-binding reagent comprises contacting the nuclei bound to the nuclei-binding reagent with a plurality of nuclei isolation particles. In some embodiments, the method comprises contacting the nuclei bound to the nuclei-binding reagent with a plurality of null particles before contacting the nuclei bound to the nuclei-binding reagent with a plurality of nuclei isolation particles. In some embodiments, the ratio of null particles to nuclei isolation particles is at least 10:1. In some embodiments, the null particles do not comprise magnetic properties. In some embodiments, the null particles do not comprise a reagent capable of specifically binding to the nuclei-binding reagent. In some embodiments, the null particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, or a combination thereof. In some embodiments, the plurality of null particles reduces aggregation of the nuclei isolation particles, prevents aggregation of the nuclei isolation particles, or both.In some embodiments, contacting the nuclei bound to the nuclei-binding reagent with a plurality of nuclei isolation particles produces a plurality of nuclei bound to the nuclei isolation particles via a reagent that can specifically bind to the nuclei-binding reagent. In some embodiments, after contacting the nuclei bound to the nuclei-binding reagent with a plurality of nuclei isolation particles, the percentage of nuclei bound to a single nuclei isolation particle is at least 90%, at least 95%, or at least 99%. In some embodiments, after contacting the nuclei bound to the nuclei-binding reagent with a plurality of nuclei isolation particles, the percentage of nuclei isolated particles that are bound to a single nuclei or that are not bound to any nuclei is at least 90%, at least 95%, or at least 99%.

[0010] In some embodiments, isolating the nuclei bound to the nuclei-binding reagent comprises isolating the nuclei isolation particles by magnetic removal, centrifugation, or any combination thereof. In some embodiments, the method comprises, prior to isolating the plurality of nuclei isolation particles, contacting the plurality of nuclei bound to the nuclei isolation particles with a liberated first epitope, wherein the liberated first epitope comprises all or a portion of the first epitope. In some embodiments, the plurality of barcodes are associated with the nuclei isolation particles. At least one barcode of the plurality of barcodes may be immobilized on the nuclei isolation particle. At least one barcode of the plurality of barcodes may be partially immobilized on the nuclei isolation particle. At least one barcode of the plurality of barcodes may be encapsulated in the nuclei isolation particle. At least one barcode of the plurality of barcodes may be partially encapsulated in the nuclei isolation particle. At least one barcode of the plurality of barcodes may not be encapsulated in the nuclei isolation particle. At least one barcode of the plurality of barcodes may be embedded in the nucleus isolation particle. At least one barcode of the plurality of barcodes may be partially embedded in the nucleus isolation particle. At least one barcode of the plurality of barcodes may not be embedded in the nucleus isolation particle.

[0011] In some embodiments, the nuclear binding reagent comprises a nuclear envelope surface component binding reagent, which may be capable of specifically binding to one or more nuclear envelope surface components.

[0012] In some embodiments, the method includes lysing the plurality of nuclei before barcoding the plurality of targets within the plurality of nuclei using the plurality of barcodes to generate a plurality of barcoded targets. In some embodiments, the method includes lysing the plurality of cells without lysing the nuclei of the plurality of cells before isolating the plurality of nuclei of the plurality of cells using a nuclei isolation composition. In some embodiments, the method includes dividing the plurality of nuclei before lysing the plurality of nuclei. In some embodiments, dividing the plurality of nuclei includes dividing the plurality of nuclei into a plurality of compartments, wherein one compartment of the plurality of compartments comprises a single nucleus from the plurality of nuclei. In some embodiments, the plurality of compartments comprise microwells of a microwell array. In some embodiments, the plurality of compartments comprise a plurality of droplets. In some embodiments, two or more compartments of the plurality of compartments comprise a single nucleus. In some embodiments, one compartment of the plurality of compartments comprises a single nucleus and a single nucleus isolated particle. In some embodiments, one compartment of the plurality of compartments comprises a single nucleus and a single barcoded particle. In some embodiments, one compartment of the plurality of compartments comprises a single nucleus, a single nucleus isolated particle, and a single barcoded particle.

[0013] In some embodiments, the method includes permeabilizing the plurality of cells (e.g., lysing their plasma membranes) prior to isolating the plurality of nuclei of the plurality of cells using a nuclei isolation composition, and depleting one or more organelles of the plurality of cells using an organelle capture composition comprising an organelle-binding reagent, where the organelle-binding reagent can specifically bind to one or more components of one or more organelles of the plurality of cells. Depleting the one or more organelles may include contacting one or more organelles of the plurality of cells with the organelle capture composition to produce one or more organelles bound to the organelle component-binding reagent. Depleting the one or more organelles includes depleting one or more organelles bound to the organelle-binding reagent using a reagent that can specifically bind to the organelle-binding reagent. The organelle-binding reagent may be associated with a second epitope, and the reagent capable of specifically binding to the organelle-binding reagent may comprise a second epitope-binding reagent.

[0014] In some embodiments, the second epitope may include biotin, a hapten, or a combination thereof. The hapten may include digoxigenin, 2,4-dinitrophenol, fluorescein, or a combination thereof. The reagent capable of specifically binding to the organelle-binding reagent may include an anti-hapten antibody. The reagent capable of specifically binding to the organelle-binding reagent may include avidin, streptavidin, neutravidin, or a combination thereof. The organelle-binding reagent may include a primary antibody capable of specifically binding to one or more components of one or more organelles of a plurality of cells, and the reagent capable of specifically binding to the organelle-binding reagent may include a secondary antibody capable of specifically binding to the primary antibody. In some embodiments, the organelle-binding reagent is associated with the organelle-capturing particle. The organelle-binding reagent may be immobilized or partially immobilized to the organelle-capturing particle. For example, the organelle-binding reagent may be reversibly, irreversibly, covalently, non-covalently, or a combination thereof associated with the organelle-capturing particle. As another example, the organelle-binding reagent may be embedded, partially embedded, not embedded, encapsulated, partially encapsulated, not encapsulated, or a combination thereof in the organelle-capturing particle.

[0015] In some embodiments, the organelle capture particle comprises an organelle capture bead. The organelle capture particle may comprise Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The organelle capture particle may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof.

[0016] In some embodiments, depleting organelles from the plurality of cells using the organelle-capturing composition may include depleting one or more organelle-capturing particles by magnetic removal, centrifugation, or any combination thereof. The organelles may include mitochondria from the plurality of cells. One or more components of one or more organelles from the plurality of cells may include ABCD3, ESR2, NOS3, ALB, HIF1A, NR3C1, ATP5A1, HK1, PGR, CASQ1, HSPA1A, PHB, CLTC, HSPD1, PLN, COX4I1, IFM1, SOD1, CPS1, LGALS3, TP53, cytochrome C oxidase, MAPT, TP5B, ERN1, MT-CO1, VDAC1, or a combination thereof. In some embodiments, the organelle-binding reagent may comprise an organelle surface component-binding reagent, one or more components of one or more organelles may comprise one or more organelle surface components, and the organelle-binding reagent may be capable of specifically binding to one or more organelle surface components. The nuclear-binding reagent may comprise a nuclear-indexing oligonucleotide, and the nuclear-indexing oligonucleotide comprises a nuclear-indexing sequence.

[0017] In some embodiments, the method includes barcoding a nucleus-indexing oligonucleotide using a plurality of barcodes to generate a plurality of barcoded nucleus-indexing oligonucleotides, and obtaining sequencing data for the plurality of barcoded nucleus-indexing oligonucleotides. Barcoding the nucleus-indexing oligonucleotides may include stochastically barcoding the nucleus-indexing oligonucleotides using a plurality of barcodes to generate a plurality of barcoded nucleus-indexing oligonucleotides. Barcoding the nucleus-indexing oligonucleotides using a plurality of barcodes may include contacting the plurality of barcodes with the nucleus-indexing oligonucleotides to generate barcodes hybridized to the nucleus-indexing oligonucleotides, and extending the barcodes hybridized to the nucleus-indexing oligonucleotides to generate a plurality of barcoded nucleus-indexing oligonucleotides. Extending the barcodes may include using a DNA polymerase to extend the barcodes to generate a plurality of barcoded nucleus-indexing oligonucleotides. Extending the barcodes may include using a reverse transcriptase to extend the barcodes to generate a plurality of barcoded nucleus-indexing oligonucleotides.

[0018] In some embodiments, the method may include amplifying a plurality of barcoded nuclear indexing oligonucleotides to obtain a plurality of barcoded nuclear indexing amplicons. Amplifying a plurality of barcoded nuclear indexing oligonucleotides may include amplifying at least a portion of the molecular label sequence and at least a portion of the nuclear indexing oligonucleotides using polymerase chain reaction (PCR). Obtaining sequencing data of the plurality of barcoded nuclear indexing oligonucleotides may include obtaining sequencing data of the plurality of barcoded nuclear indexing amplicons. Obtaining sequencing data of the plurality of barcoded nuclear indexing oligonucleotides may include sequencing at least a portion of the molecular label sequence and at least a portion of the nuclear indexing oligonucleotides.

[0019] In some embodiments, the plurality of barcodes are associated with barcoded particles. At least one barcode of the plurality of barcodes may be immobilized on the barcoded particle. At least one barcode of the plurality of barcodes may be partially immobilized on the barcoded particle. At least one barcode of the plurality of barcodes may be encapsulated in the barcoded particle. At least one barcode of the plurality of barcodes may be partially encapsulated in the barcoded particle. The barcoded particle may be disintegrable. The barcoded particle may comprise barcoded beads. The barcoded particles 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. The barcoded particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The barcoded particles may comprise disintegrable hydrogel particles.

[0020] In some embodiments, barcoding a plurality of targets using a plurality of barcodes to generate a plurality of barcoded targets includes contacting copies of the targets with target binding regions of the barcodes and reverse transcribing the plurality of targets using the plurality of barcodes to generate a plurality of barcoded targets. The method may include amplifying the plurality of barcoded targets to generate a plurality of amplified barcoded targets prior to obtaining sequencing data for the plurality of barcoded targets. Amplifying the barcoded targets to generate a plurality of amplified barcoded targets may include using polymerase chain reaction (PCR) to amplify the barcoded targets to generate a plurality of amplified barcoded targets. The method may include amplifying the plurality of amplified barcoded targets to generate a plurality of barcoded target amplicons. Amplifying the plurality of amplified barcoded targets may include amplifying a molecular beacon sequence and a sequence, or a portion thereof, of one of the plurality of targets to generate a plurality of barcoded target amplicons. Amplifying the plurality of amplified barcoded targets may include amplifying the plurality of amplified barcoded targets using polymerase chain reaction (PCR) to generate amplicons of the plurality of barcoded targets. Barcoding the plurality of targets of the cell using the plurality of barcodes to generate the plurality of barcoded targets may include stochastically barcoding the plurality of targets of the cell using a plurality of stochastic barcodes to generate the plurality of stochastically barcoded targets.

[0021] In some embodiments, each of the plurality of barcodes comprises a cell label sequence, a universal primer binding site, or any combination thereof, and the cell label sequences of at least two of the plurality of barcodes comprise the same sequence. The target binding region may comprise a poly(dT) region. At least 100 molecular label sequences of the plurality of barcodes may comprise different sequences. At least 1,000 molecular label sequences of the plurality of barcodes may comprise different sequences. At least 10,000 molecular label sequences of the plurality of barcodes may comprise different sequences. The molecular label sequences of the plurality of barcodes may comprise random sequences. The target binding region may comprise a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof. The plurality of cells comprises a tissue sample. The plurality of cells may comprise, for example, an epithelial tissue sample, frozen cells, fixed cells, formalin-fixed paraffin-embedded cells, tumor cells, fixed tumor cells, frozen tumor cells, formalin-fixed paraffin-embedded tumor cells, or a combination thereof. In some embodiments, the plurality of cells comprises one or more extranuclear cellular components. Non-limiting examples of extranuclear cellular components include mitochondria, peroxisomes, cytosol, vesicles, lysosomes, plasma membranes, chloroplasts, intramitochondrial matrix, inner mitochondrial membrane, intermembrane space, outer mitochondrial membrane, secretory vesicles, smooth endoplasmic reticulum, rough endoplasmic reticulum, Golgi apparatus, phagosomes, endosomes, exosomes, plasma membranes, microtubules, microfilaments, intermediate filaments, filopodia, ruffles, lamellipodia, sarcomeres, focal adhesions, podosomes, ribosomes, microsomes, lipid rafts, cell walls, or any combination thereof. In some embodiments, the one or more extranuclear cellular components comprise one or more undesired nucleic acid species. In some embodiments, the abundance of at least one of the one or more extranuclear cellular components is reduced by isolating multiple nuclei, depleting one or more organelles, or both. In some embodiments, the multiple cells comprise multiple targets and one or more undesired nucleic acid species. In some embodiments, the undesired nucleic acid species is derived from a non-nuclear organelle.In some embodiments, the undesired nucleic acid species include sibosomal RNA, mitochondrial RNA, and mitochondrial DNA. In some embodiments, the abundance of at least one of the one or more undesired nucleic acid species is reduced by isolating multiple nuclei, depleting one or more organelles, or both. In some embodiments, the one or more undesired nucleic acid species accounts for about 50%, about 60%, about 70%, about 80%, or more of the nucleic acid content of the multiple cells. In some embodiments, the undesired nucleic acid species represents less than 40%, less than 20%, less than 10%, or less than 5% of the amplicons of the multiple barcoded targets. In some embodiments, obtaining sequencing data for the multiple barcoded targets includes generating multiple sequencing reads. The sequencing reads of the undesired nucleic acid species may, for example, be less than 40%, less than 20%, less than 10%, or less than 5% of the total sequencing reads.

[0022] The present disclosure includes embodiments of a barcoded composition. In some embodiments, the barcoded composition includes a nucleus isolation composition comprising a nucleus binding reagent, wherein the nucleus binding reagent can specifically bind to one or more components of a nucleus, and a plurality of barcodes, each of the plurality of barcodes comprising a molecular beacon sequence and a target binding region, and the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different sequences.

[0023] In some embodiments, the composition includes a reagent capable of specifically binding to a nucleus-binding reagent. The nucleus-binding reagent may be associated with a first epitope, and the reagent capable of specifically binding to the nucleus-binding reagent may include a first epitope-binding reagent. The first epitope may include biotin, a hapten, or a combination thereof. The hapten may include digoxigenin, 2,4-dinitrophenol, fluorescein, or a combination thereof. The reagent capable of specifically binding to the nucleus-binding reagent may include an anti-hapten antibody. The reagent capable of specifically binding to the nucleus-binding reagent may include avidin, streptavidin, neutravidin, or a combination thereof.

[0024] In some embodiments, the nucleus-binding reagent comprises a primary antibody capable of specifically binding to one or more components of the nucleus, and the reagent capable of specifically binding to the nucleus-binding reagent comprises a secondary antibody capable of specifically binding to the primary antibody.

[0025] In some embodiments, the nucleic-binding reagent comprises a carbohydrate-binding reagent. The carbohydrate-binding reagent may comprise a carbohydrate-binding protein. The carbohydrate-binding protein may comprise a lectin. The lectin may comprise a mannose-binding lectin, a galactose-binding lectin, an N-acetylgalactosamine-binding lectin, an N-acetylglucosamine-binding lectin, an N-acetylneuraminic acid-binding lectin, a fucose-binding lectin, or a combination thereof. The lectin may include concanavalin A (ConA), lentil lectin (LCH), snowdrop lectin (GNA), castor bean (Ricinus communis) agglutinin (RCA), peanut agglutinin (PNA), jacalin (AIL), hairy vetch lectin (VVL), wheat germ agglutinin (WGA), elderberry lectin (SNA), Maackia amurensis leukoagglutinin (MAL), Maackia amurensis erythrocyte agglutinin (MAH), Ulex europaeus agglutinin (UEA), Aleuria aurantia lectin (AAL), or a combination thereof. The lectin may be or include an agglutinin. The agglutinin may be or include wheat germ agglutinin (WGA). The carbohydrate-binding protein may be derived from or derived from an animal, a bacterium, a virus, or a fungus. The carbohydrate-binding protein may be derived from or derived from a plant. The plant may be jack bean (Canavalia ensiformis), lentil (Lens culinaris), snowdrop (Galanthus nivalis), castor bean (Ricinus communis), peanut (Arachis hypogaea), jackfruit (Artocarpus integrifolia), vicia villosa, wheat (Triticum vulgaris), elderberry (Sambucus nigra), maackia amurensis, gorse (Ulex europaeus), aleuria aurantia, or a combination thereof.

[0026] In some embodiments, one or more components of the core include a sugar, an oligosaccharide, a polysaccharide, a derivative thereof, or a combination thereof. One or more components of the core may include a monosaccharide, a disaccharide, a polyol, a maltooligosaccharide, a non-maltooligosaccharide, a starch, a non-starch polysaccharide, a derivative thereof, or a combination thereof. One or more components of the core may include glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, maltodextrin, raffinose, stachyose, fructooligosaccharide, amylose, amylopectin, modified starch, glycogen, cellulose, hemicellulose, pectin, a hydrocolloid, a derivative thereof, or a combination thereof. One or more components of the core may be α-D-mannosyl residues, α-D-glucosyl residues, branched α-mannosidic structures of high α-mannose types, branched α-mannosidic structures of hybrid and biantennary complex N-glycans, fucosylated core regions of biantennary and triantennary complex N-glycans, α1-3 and α1-6 linked high mannose structures, Galβ1-4GalNAcβ1-R, Galβ1-3GalNAcα1-Ser / Thr, (Sia)Galβ1-3GalNAcα1-Ser / Thr, GalNAcα-Ser / Thr, GlcNAcβ1-4GlcNAcβ1-4GlcNAc, Neu5Ac (sialic acid), Neu5Acα2-6Gal(NAc)-R, Neu5Ac / Gcα2,3Galβ1,4Glc(NAc), Neu5Ac / Gcα2,3Galβ1,3(Neu5Acα2,6)GalNac, Fucα1-2Gal-R, Fucα1-2Galβ1-4(Fucα1-3 / 4)Galβ1-4GlcNAc, R2-GlcNAcβ1-4(Fucα1-6)GlcNAc-R1, derivatives thereof, or combinations thereof. One or more components of the core may include a glycoprotein, a glycolipid, or a combination thereof.

[0027] In some embodiments, the nuclei-binding reagent is associated with the nuclei isolation particles. A reagent capable of specifically binding to the nuclei-binding reagent may be associated with the nuclei isolation particles. A reagent capable of specifically binding to the nuclei-binding reagent may be immobilized or partially immobilized on the nuclei isolation particles. The nuclei isolation particles may comprise nuclei isolation beads. The nuclei isolation particles 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. The nucleus isolation particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The nucleus isolation particles may be disintegrable. The nucleus isolation particles may comprise nucleus isolation disintegrable hydrogel particles.

[0028] In some embodiments, the plurality of barcodes are associated with nucleus isolation particles. At least one barcode of the plurality of barcodes may be immobilized on the nucleus isolation particles. At least one barcode of the plurality of barcodes may be partially immobilized on the nucleus isolation particles. At least one barcode of the plurality of barcodes may be encapsulated in the nucleus isolation particles. At least one barcode of the plurality of barcodes may be partially encapsulated in the nucleus isolation particles.

[0029] In some embodiments, the one or more components of the nucleus may comprise lamin, emerin, nesprin, nurim, UNC-83, kral, ZYG-12, Kms1p, UNC-84, kraloid, SUN-1, Sad1p, LBR, MAN1, LAP1, LAP2, LINK, the nuclear pore complex, a portion thereof, or a combination thereof. In some embodiments, the nucleus binding reagent comprises a nuclear envelope surface component binding reagent, which may be capable of specifically binding to one or more nuclear envelope surface components.

[0030] In some embodiments, the composition comprises an organelle capture composition including an organelle-binding reagent, wherein the organelle-binding reagent is capable of specifically binding to one or more components of one or more organelles. The composition may include a reagent capable of specifically binding to the organelle-binding reagent. The organelle-binding reagent may be associated with a second epitope, and the reagent capable of specifically binding to the organelle-binding reagent may include a second epitope-binding reagent. The second epitope may include biotin, a hapten, or a combination thereof. The hapten may include digoxigenin, 2,4-dinitrophenol, fluorescein, or a combination thereof. The reagent capable of specifically binding to the organelle-binding reagent may include an anti-hapten antibody. The reagent capable of specifically binding to the organelle-binding reagent may include avidin, streptavidin, neutravidin, or a combination thereof. The organelle-binding reagent may include a primary antibody capable of specifically binding to one or more components of one or more organelles of the plurality of cells, and the reagent capable of specifically binding to the organelle-binding reagent may include a secondary antibody capable of specifically binding to the primary antibody.

[0031] In some embodiments, the organelle-binding reagent is associated with an organelle-capturing particle. The organelle-binding reagent may be immobilized or partially immobilized on the organelle-capturing particle. The organelle-capturing particle may comprise an organelle-capturing bead. The organelle-capturing particle 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. The organelle capture particle may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof.

[0032] In some embodiments, the organelle comprises mitochondria of a plurality of cells. One or more components of one or more organelles of the plurality of cells may comprise ABCD3, ESR2, NOS3, ALB, HIF1A, NR3C1, ATP5A1, HK1, PGR, CASQ1, HSPA1A, PHB, CLTC, HSPD1, PLN, COX4I1, IFM1, SOD1, CPS1, LGALS3, TP53, cytochrome C oxidase, MAPT, TP5B, ERN1, MT-CO1, VDAC1, or a combination thereof. In some embodiments, the organelle binding reagent comprises an organelle surface component binding reagent, wherein one or more components of one or more organelles may comprise one or more organelle surface components, and the organelle binding reagent may be capable of specifically binding to one or more organelle surface components.

[0033] In some embodiments, the nucleus-binding reagent may comprise a nucleus-indexing oligonucleotide, and the nucleus-indexing oligonucleotide comprises a nucleus-indexing sequence. The plurality of barcodes may be associated with the barcoded particle. At least one barcode of the plurality of barcodes may be immobilized on the barcoded particle. At least one barcode of the plurality of barcodes may be partially immobilized on the barcoded particle. At least one barcode of the plurality of barcodes may be encapsulated in the barcoded particle. At least one barcode of the plurality of barcodes may be partially encapsulated in the barcoded particle. The barcoded particle may be disintegrable. The barcoded particle may comprise a barcoded bead. The barcoded particles may include 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. The barcoded particles may include a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The barcoded particles may include disintegrable hydrogel particles.

[0034] In some embodiments, each of the plurality of barcodes comprises a cell labeling sequence, a binding site for a universal primer, or any combination thereof, and the cell labeling sequences of at least two barcodes of the plurality of barcodes comprise an identical sequence. The target binding region may comprise a poly(dT) region. At least 100 molecular beacon sequences of the plurality of barcodes may comprise different sequences. At least 1,000 molecular beacon sequences of the plurality of barcodes may comprise different sequences. At least 10,000 molecular beacon sequences of the plurality of barcodes may comprise different sequences. The molecular beacon sequences of the plurality of barcodes may comprise random sequences. The target binding region may comprise a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 illustrates a non-limiting exemplary barcode (e.g., a probabilistic barcode). [Figure 2] FIG. 1 illustrates a non-limiting exemplary workflow for barcoding and electronic counting (e.g., probabilistic barcoding and electronic counting). [Figure 3] 1 is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) from multiple targets. [Figure 4A-4B] 1 shows a non-limiting exemplary schematic of a nuclear barcoding workflow (e.g., probabilistic barcoding). [Figure 5A-5B] 1 shows a non-limiting, exemplary schematic diagram of a barcoding workflow (e.g., stochastic barcoding) that includes organelle (e.g., mitochondrion) removal. [Figures 6A-6F] 1 shows a non-limiting exemplary schematic of a nuclei capture and barcoding workflow. [Figure 7A-7C] 1 shows a non-limiting exemplary schematic of a nuclei capture and barcoding workflow. 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 illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein and illustrated in the 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.

[0037] 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.

[0038] Determining the number of nucleic acid or target, for example, messenger ribonucleic acid (mRNA) molecules is clinically important, for example, to identify the genes expressed in cells at different developmental stages or under different environmental conditions.However, it can be very difficult to determine the absolute number of nucleic acid molecules (for example, mRNA molecules), 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 will obtain identical molecular copies in each cycle.However, PCR can have drawbacks because each molecule replicates with a stochastic probability, and this probability varies depending on PCR cycles and gene sequence, resulting in amplification bias and inaccurate gene expression measurements.

[0039] Barcodes (e.g., stochastic barcodes) with unique molecular labels (ML, also referred to as molecular indexes (MI)) can be used to count the number of molecules. Barcodes with unique molecular labels for each cell label can be used to count the number of molecules in each cell. Non-limiting exemplary barcoding assays include Precise™ assay (Cellular Research, Inc. (Palo Alto, CA)), Resolve™ assay (Cellular Research, Inc. (Palo Alto, CA)), or Rhapsody™ assay (Becton, Dickinson and Company (Franklin Lakes, NJ)). However, these methods and techniques may introduce errors, which may overestimate cell counts if not corrected.

[0040] The Rhapsody™ assay utilizes a non-depleting pool of barcodes (e.g., stochastic barcodes) bearing a large number of unique molecular labels, e.g., 6561–65536, on poly(T) oligonucleotides to hybridize to all poly(A)-mRNA in a sample during the reverse transcription (RT) step. In addition to the molecular labels, the barcodes' cellular labels can be used to identify each single cell in each well of a microwell plate. The barcodes may contain universal PCR priming sites. During RT, target gene molecules react randomly with the barcodes. Each target molecule hybridizes to the barcode (e.g., stochastic barcode), resulting in the generation of barcoded complementary ribonucleotide acid (cDNA) molecules (e.g., stochastically barcoded cDNA molecules). After labeling, the barcoded cDNA molecules from each microwell of the microwell plate may be pooled into a single tube for PCR amplification and sequencing. The raw sequencing data can be analyzed to determine the number of barcodes with unique molecular labels.

[0041] The present disclosure includes embodiments of a method for determining the number of targets in a plurality of cells.In some embodiments, the method includes: isolating a plurality of nuclei of a plurality of cells using a nucleus isolation composition, wherein the nucleus isolation composition comprises a nucleus binding reagent, and the nucleus binding reagent can specifically bind to one or more components of the nuclei; barcoding a plurality of targets in the plurality of nuclei using a plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a molecular beacon sequence and a target binding region, and the molecular beacon sequences of at least two barcodes among the plurality of barcodes comprise different sequences; obtaining sequencing data of the plurality of barcoded targets; and using the molecular beacon sequences of the plurality of barcodes in the sequencing data to estimate the number of each of the plurality of targets in the plurality of cells.

[0042] The present disclosure includes embodiments of a barcoded composition. In some embodiments, the barcoded composition includes a nucleus isolation composition comprising a nucleus binding reagent, wherein the nucleus binding reagent can specifically bind to one or more components of a nucleus, and a plurality of barcodes, each of the plurality of barcodes comprising a molecular beacon sequence and a target binding region, and the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different sequences.

[0043] definition Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.See, for example, 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 the purposes of this disclosure, the following terms are defined below.

[0044] 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, a barcode, a stochastic barcode, or 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. The universal sequence may be a region of nucleotide sequence common to two or more nucleic acid molecules. The two or more nucleic acid molecules may 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 enable 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, enabling 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 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.

[0045] As used herein, the term "associated" or "associated with" can mean that two or more species are identifiable as being located together at a certain 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, where digital information about two or more species is stored and can be used to determine that one or more of the species were located together at a certain time. Association can also be a direct or indirect 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 synthetic particle or bead. Association can also be a covalent bond between a target and a label.

[0046] As used herein, the term "complementary" can 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 can be "partial," in which only some of the nucleotides bind, or complete, in which total complementarity exists between the single-stranded molecules. A first nucleotide sequence can 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 can 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, the terms "complement," "complementary," and "reverse complement" can be used interchangeably. It is understood from this disclosure that when a molecule is capable of hybridizing to another molecule, it may be the complement of the hybridizing molecule.

[0047] 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 probabilistic approach converts the problem of molecular counting into a series of yes / no digital questions regarding the detection of a predefined set of labels, one of locating and identifying identical molecules. As used herein, the term "label" or "labels" may refer to a nucleic acid code associated with a target in a sample. The label may be, for example, a nucleic acid label. The label may be a wholly or partially amplifiable label. The label may be a wholly or partially sequenceable label. The label may be a portion of a naturally occurring nucleic acid that can be identified as distinct. The label may be a known sequence. The label may comprise a junction of a nucleic acid sequence, e.g., a junction of a naturally occurring and non-natural sequence. As used herein, the term "label" may be used interchangeably with the terms "index," "tag," or "label tag." The label may 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 stochastic barcodes composed of a large number of different labels. The non-depletion reservoir can contain a large number of different stochastic barcodes, so that when the non-depletion reservoir is associated with a pool of targets, each target is likely to associate with a unique stochastic 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 then analyzing the number of detected stochastic barcodes allows for the 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 stochastic 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 contain 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. Nucleosides may be base-sugar combinations. The base portion of a nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides may be nucleosides 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. When forming nucleic acids, the phosphate group may covalently link adjacent nucleosides to each other 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 produce fully or partially double-stranded compounds. Within nucleic acids, the phosphate groups may generally 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 can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, e.g., 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, e.g., 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 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 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 linked 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 linked to the aza nitrogen atoms of the amide portion of the backbone.

[0054] Nucleic acid can include morpholino backbone structure.For example, nucleic acid can include 6-membered morpholino ring instead of ribose ring.In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkage can replace phosphodiester linkage.

[0055] Nucleic acids can contain linked morpholino units (i.e., 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 termed 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.

[0056] 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, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Modified nucleobases 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).

[0057] 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 term "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.

[0058] As used herein, the term "solid support" may refer to a solid or semi-solid surface to which multiple stochastic barcodes can be attached. A solid support may include 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 include 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 plurality of solid supports spaced apart in an array may not include a substrate. A solid support may also be a particle, such as a synthetic particle or bead. A solid support may be referred to as a "substrate." A substrate may be any type of solid support. A substrate may refer to a continuous solid or semi-solid surface on which the methods of the present disclosure can be performed. A substrate may refer to, for example, an array, a cartridge, a chip, a device, and a slide. As used herein, the term "spatial label" may refer to a label that can be associated with a location in space.

[0059] As used herein, the term "stochastic barcode" may refer to a polynucleotide sequence containing a label. 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 in 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.

[0060] As used herein, the term "gene-specific stochastic barcode" may refer to a polynucleotide sequence that includes a label and a gene-specific target binding region. The stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. The stochastic barcode may be used to quantify a target in a sample. The stochastic barcode may be used to control errors that may occur after the label is associated with the target. For example, the stochastic barcode may be used to evaluate amplification or sequencing errors. The stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target.

[0061] As used herein, the term "probabilistic barcoding" can refer to random labeling (e.g., barcoding) of nucleic acids. Probabilistic barcoding can utilize a Poisson recursive strategy to associate labels and quantify the labels associated with targets. As used herein, the term "probabilistic barcoding" can be used interchangeably with "gene-specific probabilistic barcoding." As used herein, the term "target" may refer to a composition that can be associated with a stochastic barcode. Exemplary targets suitable for analysis by the disclosed methods, devices, and systems 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. In some embodiments, targets are lipids.

[0062] As used herein, the term "reverse transcriptase" can refer to a group of enzymes that have reverse transcriptase activity (i.e., 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).

[0063]

[0003] A system and method for identifying a single cellular marker is disclosed herein. In some embodiments, the method includes: (a) stochastically barcoding a plurality of targets in a cell sample using a plurality of stochastic barcodes to generate a plurality of stochastically barcoded targets, wherein each of the plurality of stochastic barcodes includes a cellular marker and a molecular marker; (b) obtaining sequencing data for the plurality of stochastically barcoded targets; (c) determining the number of molecular markers having distinct sequences associated with each of the cellular markers of the plurality of stochastic barcodes; (d) determining the rank of each of the cellular markers of the plurality of stochastic barcodes based on the number of molecular markers having distinct sequences associated with each of the cellular markers; and (e) ( (c) generating a cumulative sum plot based on the number of molecular labels having distinct sequences associated with each of the cellular markers determined in (c) and the rank of each of the cellular markers determined in (d); (f) generating a second derivative plot of the cumulative sum plot; (g) determining a minimum value of the second derivative plot of the cumulative sum plot, wherein the minimum value of the second derivative plot corresponds to a threshold value for the cellular marker; and (h) identifying each of the cellular markers as a signal cellular marker or a noise cellular marker based on the number of molecular labels having distinct sequences associated with each of the cellular markers determined in (c) and the cellular marker threshold value determined in (g).

[0064] Barcode Barcoding, e.g., stochastic barcoding, is described, for example, in U.S. Patent Application Publication No. US20150299784, International Publication No. WO2015031691, and Fu et al., Proc Natl Acad Sci USA 2011 May 31;108(22):9026-31 and Fan et al., Science (2015) 347(6222):1258367, the contents of each of which are incorporated herein in their entirety. In some embodiments, the barcodes disclosed herein may be stochastic 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, for example, mRNA species that include mRNA molecules with identical or nearly identical sequences. A barcode may be referred to as a stochastic barcode when 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 the stochastic barcode may be referred to as molecular labels.

[0065] 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 108. 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. In some cases, the labels of the barcode (e.g., universal label, dimensional label, spatial label, cellular label, and barcode sequence) can be spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides apart.

[0066] 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 within 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 the sequence dataset of the labeled target nucleic acid molecule (described in more detail below) can enable amplification or sequencing errors to be detected or corrected. 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 any two of these values, or a length of approximately these values ​​or such a number or range of nucleotides. In some embodiments, nucleic acid subsequences of other lengths may be used to create error-correcting codes.

[0067] 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 containing a poly(A) tail, or any combination thereof. In some embodiments, the multiple targets may include deoxyribonucleic acid (DNA).

[0068] 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 one or more spacers. 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 length. In some embodiments, none of the labels of the barcode are separated by a spacer.

[0069] 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 particles (e.g., synthetic particles, e.g., 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 the 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. Universal label can comprise a sequence that can be used for barcode or barcode region extension.Universal label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or any two of these values ​​or ranges, or approximately these values ​​or ranges of nucleotides in length.For example, universal label can comprise at least about 10 nucleotides.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 nucleotide may be part of the universal label sequence to allow for cleavage of the barcode from the support.

[0070] 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 stochastically 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 stochastically barcoded. For example, a cell population may be stochastically 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 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 a therapeutic agent. Changes in the copy number of distinct targets can indicate the sample's response to the drug and / or therapeutic agent.

[0071] 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 may be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Dimensional labels may 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.

[0072] 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., synthetic particles, e.g., beads), but may be different for different solid supports (e.g., synthetic particles). 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.

[0073] Multiple solid supports (e.g., synthetic particles) are 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, 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. 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.

[0074] spatial sign The barcode may include one or more spatial labels. In some embodiments, the spatial label may include a nucleic acid sequence that provides information about the spatial orientation of the target molecule associated with the barcode. The spatial label may be associated with a coordinate in the sample. The coordinate may be a fixed coordinate. For example, the coordinate may be fixed relative to a substrate. The spatial label may refer to 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, a fluorescent property, a radioactive property, or a structure with a unique size or shape. The spatial label may be associated with a physical compartment (e.g., a well, a container, a microsphere, a tube, a microcapsule, or a droplet). In some embodiments, multiple spatial labels are used together to encode one or more locations in space.

[0075] Spatial labels may be the same for all barcodes attached to a given solid support (e.g., synthetic particles, e.g., beads), but may be different for different solid supports (e.g., synthetic particles). 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.

[0076] A plurality of solid supports (e.g., synthetic particles, e.g., beads) are 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 from about 5 to about 200 nucleotides. Spatial markers can comprise from about 10 to about 150 nucleotides. Spatial markers can comprise from about 20 to about 125 nucleotides in length.

[0077] cell labeling A 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 synthetic particle, e.g., a bead), but is different for different solid supports (e.g., a synthetic particle). In some embodiments, the percentage of barcodes containing the same cell 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 approximately these values ​​or such number or range. In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%, or approximately these values. 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.

[0078] Multiple solid supports (e.g., synthetic particles) are 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 from about 5 to about 200 nucleotides. As another example, a cell marker can comprise from about 10 to about 150 nucleotides. As yet another example, a cell marker can comprise from about 20 to about 125 nucleotides in length.

[0079] 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). In some embodiments, a diverse set of barcode sequences is attached to a given solid support (e.g., a synthetic particle, 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 approximately, any two of these values. For example, the plurality of barcodes may include about 6561 barcode sequences with distinct sequences. As another example, the plurality of barcodes may include about 65536 barcode sequences 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 barcode sequences. The unique molecular beacon sequences can be attached to a given solid support (e.g., a synthetic particle).

[0080] A barcode 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. A barcode 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] molecular label The stochastic barcode may include one or more molecular labels. The molecular label may include a barcode sequence. In some embodiments, the molecular label may include a nucleic acid sequence that provides information about the specific type of target nucleic acid species that hybridizes to the stochastic barcode. The molecular label includes a nucleic acid sequence that provides a counter for the specific occurrence of the target nucleic acid species that hybridizes to the stochastic barcode (e.g., target binding region). In some embodiments, a diverse set of molecular labels is attached to a given solid support (e.g., a synthetic particle, 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 of, or approximately these values, numbers, or ranges of unique molecular label sequences. For example, the plurality of stochastic barcodes may include about 6561 molecular labels with distinct sequences. As another example, the plurality of stochastic barcodes may include about 65536 molecular labels with distinct sequences. In some embodiments, 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 9There can be a number of unique molecular label sequences. Stochastic barcoding with unique molecular label sequences can be attached to a given solid support (e.g., a synthetic particle).

[0082] For probabilistic barcoding using multiple probabilistic barcodes, 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 can be approximately these values ​​or such numbers or ranges. The targets can be mRNA species that include mRNA molecules with identical or nearly identical sequences. In some embodiments, the ratio of the number of different molecular label 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.

[0083] 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.

[0084] 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, e.g., a cellular nucleic acid to be analyzed). For example, the target binding region may hybridize to a target nucleic acid at a specific gene sequence. In some embodiments, the target binding region may include 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.

[0085] In some embodiments, the target binding region may include 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 include a random multimer sequence or an oligo(dT) sequence that hybridizes to the 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 synthetic particle (e.g., a bead). In some embodiments, the target binding regions of multiple barcodes bound to a given synthetic particle may include two or more different target binding sequences. A target binding region can be 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. A target binding region can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more nucleotides in length.

[0086] In some embodiments, the target binding region can comprise an oligo(dT) that can hybridize with mRNA containing polyadenylated ends. The target binding region can be gene-specific. For example, the target binding region can be configured to hybridize with a specific region of the target. The target binding region can 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 of nucleotides 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 to 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.

[0087] In some embodiments, the barcode does not include a target binding region. In some embodiments, the barcode includes a region corresponding to a target binding region having a sequence that has low binding affinity (e.g., does not bind) to some, most, substantially all, or all mRNA molecules in one or more cells of the sample. For example, the barcode may include a region corresponding to a target binding region that may have a sequence that does not bind to the mRNA molecule of interest. If the target binding region of a first barcode includes an oligo(dT) sequence that can hybridize to the poly(A) tail of an mRNA molecule, the corresponding region of a second barcode may include, for example, a sequence that is dissimilar or substantially dissimilar to the poly(dT) sequence. If the target binding region of a first barcode includes a target binding region having a sequence that can specifically hybridize to a particular gene sequence, the corresponding region of a second barcode may include, for example, a sequence that is dissimilar or substantially dissimilar to the target binding region.

[0088] Orientation Characteristics Barcodes may include one or more orientation properties that can be used to orient (e.g., align) the barcodes. Barcodes may include moieties 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, orientation properties can be used to develop a known map of 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, barcodes with self-assembly orientation properties may self-assemble into a specific orientation (e.g., nucleic acid nanostructures) upon activation.

[0089] affinity properties A barcode may include one or more affinity features. For example, a spatial label may 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 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. 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.

[0090] 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.

[0091] Universal Adapter Primer A barcode can include one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific stochastic barcode, can include a universal adapter primer. The universal adapter primer can refer to a universal nucleotide sequence across all barcodes. The universal adapter primer can be used to construct a gene-specific barcode. The universal adapter primer can 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.

[0092] Linker When a barcode includes more than one type of label (e.g., more than one cell 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 at most 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 can be used to facilitate the synthesis of the barcode. The linker label may include an error-correcting (e.g., Hamming) code.

[0093] 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 particle or 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.

[0094] The synthetic particles 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 synthetic particles can include materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof.

[0095] In some embodiments, the synthetic particles can be polymer particles, such as deformable particles or gel particles (e.g., gel beads from 10X Genomics (San Francisco, CA)) functionalized with barcodes or stochastic barcodes. In some implementations, the gel particles can include one or more polymer-based gels. Gel particles can be generated, for example, by encapsulating one or more polymer precursors in droplets. Gel particles can be generated when the polymer precursors are exposed to an accelerator (e.g., tetramethylethylenediamine (TEMED)). In some embodiments, the particles may be degradable. For example, polymer particles or beads may dissolve, melt, or decompose under desired conditions. The desired conditions may include environmental conditions. The desired conditions may result in the dissolution, melt, or decomposition of the polymer particles in a controlled manner. Gel particles may dissolve, melt, or decompose due to chemical, physical, biological, thermal, magnetic, electrical, or optical stimuli, or any combination thereof.

[0096] Reagents, e.g., oligonucleotide barcodes, may be linked / immobilized to the interior surface of synthetic particles (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 gel particles or any other microcapsules described herein. The particles may be, for example, gel beads. Association (e.g., linkage or 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, association (e.g., linkage or immobilization) of reagents to particles or any other solid support (e.g., microcapsules) 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 may be cleaved, liberating 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 particle via a disulfide bond, exposing the disulfide bond to a reducing agent can cleave the disulfide bond and release the oligonucleotide barcode from the particle. The labile moiety can be introduced as part of the gel particle, bead, or microcapsule, as part of a chemical linker connecting a reagent to the gel bead or microcapsule, and / or as part of the reagent. In some embodiments, at least one barcode of the plurality of barcodes can be immobilized to the particle, partially immobilized to the particle, encapsulated in the particle, partially encapsulated in the particle, or any combination thereof.

[0097] In some embodiments, the particles (e.g., gel beads) may 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) (PTHF), poly(hexylviologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), and poly(lactic-co-glycolic acid) (PLGA).

[0098] A number of chemical stimuli can be used to trigger particle disintegration, dissolution, or decomposition. Examples of these chemical changes include, but are not limited to, pH-mediated changes to the particle wall, particle wall disintegration via chemical cleavage of cross-links, triggering particle wall depolymerization, and particle wall switching reactions. Bulk changes can also be used to trigger particle disintegration.

[0099] Bulk or physical changes to microcapsules or particles through various stimuli also offer many advantages in designing capsules for releasing reagents. Bulk or physical changes occur on a macroscopic scale, with particle rupture being the result of mechanical-physical forces induced by the stimulus. These processes can include, but are not limited to, pressure-induced rupture, particle wall melting, or changes in particle wall porosity.

[0100] Biological stimuli can also be used to trigger particle 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, particles 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 particle's contents. In other cases, the protease can be heat-activated. In another example, particles contain a shell wall containing cellulose. Addition of the hydrolytic enzyme chitosan serves as a biological trigger to cleave the cellulose bonds, depolymerize the shell wall, and release its contents.

[0101] Particles can also be induced to release their contents upon application of a thermal stimulus. A change in temperature can cause various changes in the particles. A change in heat can cause the particle to melt, causing the particle wall to collapse. In other cases, heat can increase the internal pressure of the particle's internal components, causing the particle to collapse or explode. In yet other cases, heat can transform the particle into a compressed, dehydrated state. Heat can also act on a thermosensitive polymer within the particle's wall, causing the particle to collapse. By including magnetic nanoparticles in the particle walls of microcapsules, it is possible to trigger particle collapse and guide the particles in an array. The device of the present disclosure can include magnetic particles for any purpose. In one example, Fe3O4 nanoparticles are incorporated into polyelectrolyte-containing particles to trigger collapse in the presence of an oscillating magnetic field stimulus.

[0102] Particles 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 particles can trigger both particle disintegration and other functions, such as alignment in an electric field, electrical conduction, or redox reactions. In one example, particles containing electrically sensitive materials are aligned in an electric field to control the release of internal reagents. In another example, the electric field can induce redox reactions within the particle wall itself, which can increase porosity. Light stimulation can also be used to disrupt particles. 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 particle wall. In yet another example, photoswitchable materials, such as azobenzene groups, can be incorporated into the particle 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 particle walls that can collapse or become more porous upon application of a light trigger.

[0103] 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, particles can be introduced into multiple microwells of the microwell array in block 212. Each microwell can contain one particle. The particles can include multiple barcodes. The barcodes can include 5' amine regions attached to the particles. The barcodes can include a universal label, a barcode sequence (e.g., a molecular label), a target binding region, or any combination thereof.

[0104] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., a particle or 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 the solid support may comprise barcode sequences of different sequences. In some embodiments, a percentage of the barcodes associated with the solid support comprise the same cell marker. 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.

[0105] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., a particle or bead). In some embodiments, stochastically 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 particles 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 barcode may be an individual nucleotide. The barcode may be associated with a substrate.

[0106] 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 particle, e.g., a bead. The particle may comprise one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar structures that can immobilize nucleic acids (e.g., covalently or non-covalently). The particle may be composed of, for example, plastic, ceramic, metal, polymeric material, or any combination thereof. The particle may be or comprise a separate particle that is spherical (e.g., a microsphere), or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. In some embodiments, the particle may be non-spherical in shape.

[0107] The particles 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 particle (e.g., the particle to which the label is attached) is a hydrogel bead. In some embodiments, the particle comprises a hydrogel.

[0108] 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), 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 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 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, at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more of the particles contain oligonucleotides with the same cell sequence. In some embodiments, none of the particles in the plurality have the same cell-labeling sequence.

[0109] 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 may contain 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 may contain different barcode sequences. Some embodiments provide a plurality of particles comprising barcodes. In some embodiments, the ratio of occurrence (or copy or number) of the target to be labeled to the different barcode sequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or more. In some embodiments, each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both. The particle can be, for example, a nanoparticle or a microparticle.

[0110] The size of the particles can vary. For example, the diameter of the particles can range from 0.1 micrometers to 50 micrometers. In some embodiments, the diameter of the particles 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.

[0111] The diameter of a particle may be related to the diameter of a well in a substrate. In some embodiments, the diameter of a particle may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between or about any two of these values, longer or shorter than the diameter of the well. The diameter of a particle may be related to the diameter of a cell (e.g., a single cell surrounded by a well in a substrate). In some embodiments, the diameter of a particle may be at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the well. The diameter of a particle may be related to the diameter of a cell (e.g., a single cell surrounded by a well in a substrate). In some embodiments, the diameter of a particle may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or range between or about any two of these values, or any such number or range, longer or shorter than the diameter of a cell. In some embodiments, the diameter of a particle may be at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% longer or shorter than the diameter of a cell. The particles may be bound and / or embedded in a substrate. The particles may be bound and / or embedded in a gel, hydrogel, polymer, and / or matrix. The spatial location of the particles within the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using spatial labels present in barcodes on the particles, which can serve as location addresses.

[0112] Examples of particles 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.

[0113] The particles 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 particles can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The particles can be identifiable. For example, the particles can be imaged using a camera. The particles can have a detectable code associated with them. For example, the particles can include a barcode. The particles can change size, for example, due to swelling in an organic or inorganic solution. The particles can be hydrophobic. The particles can be hydrophilic. The particles can be biocompatible.

[0114] The solid support (e.g., particle) can be visualized. The solid support can include a visualization tag (e.g., a fluorescent dye). The solid support (e.g., particle) can be etched with an identifier (e.g., a number). The identifier can be visualized through imaging of the particle. A solid support can comprise an insoluble, semi-soluble, or insoluble material. A solid support can be referred to as "functionalized" if it contains a linker, scaffold, building block, or other reactive moiety attached thereto, but can be "non-functionalized" if it lacks such a reactive moiety attached thereto. A solid support can be freely used in solution, for example, in a microtiter well format, in a flow-through format, for example, in a column, or in a dipstick.

[0115] 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, silicon, and copper), glass support, plastic support, silicon support, chip, filter, membrane, microwell plate, slide, multiwell plate, or plastic material (e.g., made of 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 silicon wafer), a wafer with holes or without a filter bottom, or particles (e.g., synthetic particles, e.g., beads).

[0116] 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. The solid support may be a biomolecule. For example, the solid support may be or include a nucleic acid, a protein, an antibody, a histone, a cellular compartment, a lipid, a carbohydrate, etc. A biomolecule solid support may be amplified, translated, transcribed, degraded, and / or modified (e.g., pegylated, sumoylated, acetylated, methylated). A biomolecule solid support may provide spatial and temporal information in addition to the spatial label attached to the biomolecule. For example, a biomolecule may contain a first conformation when unmodified, but may change to a second conformation when modified. The different conformations may expose the barcodes (e.g., stochastic barcodes) of the present disclosure to the target. For example, a biomolecule may contain a barcode that is inaccessible due to biomolecule folding. When the biomolecule is modified (e.g., acetylated), the biomolecule may change conformation to expose the barcode. The timing of the modification may provide another time dimension to the barcoding method of the present disclosure.

[0117] In some embodiments, the biomolecule comprising the barcode reagent of the present disclosure may be located in the cytoplasm of a cell. Upon activation, the biomolecule may translocate to the nucleus, where barcoding may occur. In this way, modification of the biomolecule may encode additional spatiotemporal information about the target identified by the barcode.

[0118] 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 the barcodes and stochastic barcodes of the present disclosure. A substrate may, for example, include multiple 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 with 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 may incorporate a single cell and a single solid support (e.g., a particle). A microwell may include a combinatorial barcode reagent of the present disclosure.

[0119] Synthesis of barcodes on solid supports Barcodes (e.g., stochastic barcodes) can be synthesized on solid supports (e.g., particles, e.g., synthetic particles or beads). Pre-synthesized barcodes (e.g., containing a 5' amine that can be linked to a solid support) can be attached to solid supports (e.g., particles) by any of a variety of immobilization techniques involving functional group pairs on the solid support and the barcode. The barcodes can include functional groups. The solid support (e.g., particles) can include functional groups. The functional groups on the barcodes and solid supports can include, for example, biotin, streptavidin, primary amines, carboxyl, hydroxyl, aldehyde, ketone, and any combination thereof. The barcodes (e.g., stochastic barcodes) can be tethered to solid supports by, for example, linking the 5' amino group on the barcode to a carboxyl group on a functionalized solid support (e.g., using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). Residual unlinked barcodes can be removed from the reaction mixture by performing multiple rinsing steps. In some embodiments, the barcode and solid support are indirectly linked through a linker molecule (e.g., a short, functionalized hydrocarbon or polyethylene oxide molecule) using similar linking chemistries. The linker may be a cleavable linker, such as an acid-labile linker or a photocleavable linker.

[0120] Barcodes (e.g., stochastic barcodes) can be synthesized on a solid support (e.g., a particle) using any of several solid-phase oligonucleotide synthesis techniques, such as phosphodiester synthesis, phosphotriester synthesis, phosphite triester synthesis, and phosphoramidite synthesis. Single nucleotides can be linked stepwise to incrementally tethered barcodes. Short, pre-synthesized sequences (or blocks) of several oligonucleotides can be linked to incrementally tethered barcodes.

[0121] Barcodes (e.g., stochastic barcodes) can be synthesized by interspersing stepwise or block ligation reactions with one or more rounds of split-pool synthesis, in which the entire pool of synthesized particles is divided into several smaller individual pools, each of which is then subjected to a different ligation reaction. The individual pools are then recombined and mixed to randomize the barcode sequences, increasing the number of barcodes across the entire pool of particles. Split-pool synthesis is an example of a combinatorial synthesis process that maximizes the number of chemical compounds synthesized using the minimum number of chemical ligation steps. The potential diversity of the resulting compound library is determined by the number of unique building blocks (e.g., nucleotides) available for each ligation step and the number of ligation steps used to create the library. For example, a split-pool synthesis involving 10 ligations using four different nucleotides in each step can generate 4 10 This results in 1,048,576 unique nucleotide sequences. In some embodiments, split pool synthesis can be performed using enzymatic methods such as polymerase extension or ligation reactions rather than chemical linkage. For example, in each round of split pool polymerase extension reaction, a semi-random primer, e.g., 5'-(M) , is attached to the 3' end of the barcode tethered to the particle in a given pool. k -(X) i -(N) j -3'(in the formula, (X) i is a random sequence of nucleotides that is i nucleotides long ((X) i a set of primers containing all possible combinations of j is a particular nucleotide (or a series of j nucleotides), and (M) k The 5' ends of a set of primers having the structure (where k is a specific nucleotide (or series of k nucleotides)) can be hybridized, where a different deoxyribonucleotide triphosphate (dNTP) is added to each pool and incorporated into the tethered oligonucleotide by a polymerase.

[0122] Barcoding methods The present disclosure provides a method for estimating the number of distinct targets in a sample. In some embodiments, barcoding a plurality of targets comprises hybridizing a plurality of barcodes to a plurality of targets to generate barcoded targets (e.g., stochastically barcoded targets). Barcoding a 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).

[0123] Contacting the sample with the barcode The present disclosure provides a method for contacting a sample (e.g., cells) with a substrate of the present disclosure. For example, a sample including a thin section of cells, an organ, or a 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 placed 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.

[0124] Cell lysis After the cells and barcodes are separated, the cells can be lysed to release the target molecule. Cell lysis can be achieved by any of a variety of means, for example, by chemical or biochemical means, by osmotic shock, or by thermal lysis, mechanical lysis, 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 between the target and the barcode, the diffusion rate of the target molecule can be changed, for example, by lowering the temperature and / or increasing the viscosity of the lysate.

[0125] 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 to promote lysis of the sample and hybridization of the sample's targets to the substrate.

[0126] 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, or up to about, 0.01M, 0.05M, 0.1M, 0.5M, or 1M Tris-HCl, or more or less. The lysis buffer can include about 0.01M, 0.05M, 0.1M, 0.5M, or 1M 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 contain 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 contain 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 surfactant in the lysis buffer can 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 surfactant in the lysis buffer is about 1% Li-dodecyl sulfate.The time used in the lysis method can depend on the amount of surfactant used.In some embodiments, the more surfactant used, the less time is required for lysis.The lysis buffer may include a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent in the lysis buffer may be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM, or higher. The concentration of the chelating agent in the lysis buffer may be up to about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM, or higher. 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 agent in the lysis buffer may be at least about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM, or higher. The concentration of the reducing reagent in the lysis buffer may be up to about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM or higher. 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.

[0127] 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 can 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 can contain up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules.

[0128] Binding of barcodes to target nucleic acid molecules After cell lysis and release of the 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 particles. For example, a single-stranded nucleotide fragment can hybridize to the target binding region of the barcode.

[0129] 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. 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. The labeled targets can be pooled, e.g., by collecting particles with bound barcodes and / or target-barcode molecules.

[0130] Solid support-based collection of bound target-barcode molecules can be recovered using magnetic particles 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.

[0131] Nucleic acid extension reactions (e.g., reverse transcription) The present disclosure provides methods for generating target-barcode conjugates using a nucleic acid extension reaction, e.g., reverse transcription (e.g., block 224 of Figure 2). 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.

[0132] In some embodiments, reverse transcription of the labeled RNA molecule can occur by the addition of a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, 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. 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.

[0133] 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 (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).

[0134] In some embodiments, amplification can be carried out using polymerase chain reaction (PCR).As used herein, PCR can refer to the reaction for amplifying specific DNA sequences in vitro by simultaneous primer extension of complementary strands of DNA.As used herein, PCR can encompass derivatives of reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR and assembly PCR.

[0135] 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) method, 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.

[0136] 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 a labeled amplicon (e.g., a stochastically labeled amplicon). The labeled 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 label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicon may be a single-stranded molecule. The single-stranded molecule may comprise DNA, RNA, or a combination thereof. The nucleic acids of the present disclosure may include synthetic or modified nucleic acids.

[0137] 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.

[0138] 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 fixed panel of primers. The one or more primers may comprise 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 gene-specific primers.

[0139] 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.

[0140] Any amplification scheme can be used in the disclosed method. For example, in one scheme, the first PCR can amplify molecules bound to particles (e.g., beads) using a gene-specific primer and a primer for the sequence of universal Illumina sequencing primer 1. The 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. The 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 markers and barcode sequences (e.g., molecular markers) on read 1, genes on read 2, and sample indexes on index 1 read.

[0141] 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.

[0142] 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 being 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.

[0143] 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. During 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 obtain two single-stranded nucleic acid molecules, each with one end bound to the particle surface through the first and second primers and the other end unbound. During the annealing and extension step of this second cycle, each strand can hybridize to a previously unused complementary primer on the same particle to form a new single-stranded bridge. The two previously unused primers hybridized at this point extend to convert the two new bridges into double-stranded bridges.

[0144] 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.

[0145] In some embodiments, the amplification of the labeled nucleic acid comprises a non-PCR-based method. Examples of non-PCR-based methods include, but are not limited to, multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-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), Qβ replicase (Qβ) method, 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 / or branched extension amplification (RAM).

[0146] 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 a 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 acid of the present invention may comprise a synthetic nucleic acid or a modified nucleic acid.

[0147] In some embodiments, the method includes 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 method includes performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0148] 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.

[0149] 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.

[0150] Performing one or more amplification reactions may include 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 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 plurality of labeled nucleic acids. The one or more primers may comprise a fixed panel of primers. The one or more primers may comprise 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 attached to the array of the present disclosure.

[0151] In some embodiments, barcoding (e.g., stochastically barcoding) a plurality of targets in a sample further comprises generating an indexed library of barcoded fragments. The barcode sequences of different barcodes (e.g., molecular labels of different stochastic barcodes) may be different from each other. Generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) comprises 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 any two of these values, or by approximately, or by at least, or by at most, 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 of which comprises 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.

[0152] Stochastic barcoding can use nucleic acid barcodes or tags to label individual nucleic acid (e.g., DNA or RNA) molecules. In some embodiments, this involves adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can minimize PCR amplification bias. For example, adapters can be added for sequencing using next-generation sequencing (NGS). For example, sequencing results can be used to determine the cellular label, barcode sequence (e.g., molecular label), and nucleotide fragment sequence of one or more copies of the target in block 232 of FIG. 2.

[0153] 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., mRNA. As shown in step 1, a reverse transcription process can encode each mRNA molecule with a unique barcode sequence (e.g., molecular label), a cell label, and a universal PCR site. For example, 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 RNA molecule 302. Each of barcodes 310 can include a target binding region, e.g., a poly(dT) region 312, a barcode sequence or molecular label 314, and a universal PCR region 316.

[0154] In some embodiments, the cell label may comprise 3 to 20 nucleotides. In some embodiments, the barcode sequence (e.g., molecular label) 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.

[0155] 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 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 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.

[0156] 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 numbers or ranges of nucleotides in length. 2 , 10 3 , 10 4 , 10 5 , 106 , 10 7 , 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 up to these values ​​or such number or range of barcodes or stochastic barcodes 310. Also, the set of barcodes or stochastic barcodes 310 may 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.

[0157] 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 PCR primer pool 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 in a single reaction volume using 10, 20, 40, 50, 70, 80, 90, 10, 25, 30, 35 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 1012 , 10 13 , 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 of 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.

[0158] 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. For example, nested PCR-labeled amplicon 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. 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, or may contain approximately, or at least, or at most, these values ​​or numbers or ranges of different nested PCR primers 330. Nested PCR primers 332 may contain adaptors 334 and hybridize to regions within cDNA portion 306" of 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.

[0159] 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 primers 340 and 342 can be PCR amplification primers. One or more primers 340 and 342 can be sequencing primers. One or more adapters 334 and 336 can be used for further amplification of adapter-labeled amplicons 338. One or more adapters 334 and 336 can be used for sequencing of adapter-labeled amplicons 338. Primer 342 can contain a plate index 344, allowing 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).

[0160] Methods and compositions for barcoding nuclei The present disclosure includes methods, compositions, and kits for performing both single nuclei capture and barcoding (e.g., in a single step). Sequencing of single nuclei can be used for single-cell transcriptome analysis (or other single-cell omics or multi-omics analyses, such as proteomics analysis to determine the protein expression profile of a single cell), while minimizing (e.g., eliminating) the need to perform or prepare a single-cell suspension. Preparing a single-cell suspension can be technically challenging, for example, with certain sample preparations, such as frozen tissue. The method includes a nuclei isolation step. For example, a nuclear envelope-specific antibody, such as anti-LAMIN, can be used to separate nuclei from other intracellular components.

[0161] In some embodiments, these antibodies may be associated (e.g., conjugated) with an epitope (e.g., a strong epitope including biotin, digoxigenin, or fluorescein) or a molecular barcode representing that particular antibody, and / or both. The associated epitope (e.g., a conjugated epitope) may enable a rapid isolation step of nuclei without the use of an ultracentrifugation step. For example, when multiple samples are pooled together to perform a single downstream next-generation sequencing (NGS) library preparation procedure, sample indexing can be performed using the associated barcode (e.g., a conjugated barcode). In some embodiments, sample indexing may include sample indexing for single nuclei. In some embodiments, sample indexing may include sample indexing for single nuclei and single cells.

[0162] In some embodiments, the antibody barcode may be or include a nucleotide barcode. For example, the nucleotide barcode may be at least 10 base pairs in length. The nucleotide barcode may be or include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), or other synthetic nucleotides compatible with reverse transcription or other DNA polymerization steps. The nucleotide barcode may also include a 3' poly(dA) tail to mimic endogenous cellular mRNA and create a barcode compatible with downstream library preparation. The barcode sequence information can then be demultiplexed after sequencing with each nucleus to determine which sample the nucleus originates from. Barcoded antibodies and their uses, such as cellular sample indexing using barcoded antibodies, are described in U.S. Patent Application Publication No. 2018 / 0088112 and U.S. Patent Application No. 15 / 937,713, the contents of each of which are incorporated by reference in their entirety.

[0163] How to barcode nuclei Figures 4A-4B show non-limiting, exemplary schematic diagrams of a nuclear barcoding workflow (e.g., stochastic barcoding). Cell lysis can be performed using a homogenizer (e.g., a Dounce homogenizer), detergent, or enzymatic methods. For example, nuclei can be captured by an antibody specific for a nuclear envelope protein (e.g., LAMIN) conjugated to an epitope (e.g., a strong epitope such as biotin, digoxigenin (DIG), and fluorescein) and, optionally, a barcode oligonucleotide to label the nuclei. A secondary antibody against the epitope can be used to pull down (e.g., separate from the cytoplasmic fragment) the nuclei for purification, followed by RNA / DNA capture using a single-cell analysis assay (e.g., the Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ)).

[0164] The present disclosure includes embodiments of a method 400 for determining the number of targets in a plurality of cells (e.g., cells 404 shown in Figures 4A-4B). Method 400 may include cell lysis, separation and isolation of nuclei, nuclei lysis, and barcoding for single-cell analysis (e.g., transcriptome or proteome analysis of single cells).

[0165] In some embodiments, method 400 includes isolating multiple nuclei from multiple cells using a nuclei isolation composition. The isolated multiple nuclei may include nuclei 408 including nuclear envelopes 412, as shown in FIGS. 4A-4B. Single-cell methods, such as the Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ), can be used to analyze the content (e.g., mRNA content or protein content) of the isolated nuclei, e.g., mRNA molecules 416 in nuclei 408. The nuclei isolation composition may include a nuclei-binding reagent, which may be capable of specifically binding to one or more components of a nucleus (e.g., nuclear envelope protein 420 on the nuclear envelope 412 of cell nucleus 408 of cell 404 shown in FIGS. 4A-4B).

[0166] The number of isolated nuclei may vary in different implementations. In some embodiments, the number of isolated nuclei 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, 111, 112, 113, 114, 115, 116, 117, 0, 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 3, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 900 0, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 10 7 , 10 8 , 10 9 , 1010 , 10 11 , 10 12 or a number or range between any two of these values, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 , 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 80 00, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12In some embodiments, the number of isolated nuclei is 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, 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, 120, 7, 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 1, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 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, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 10 7 , 10 8 , 10 9 , 10 10 , 10 11 Or 10 12 may be.

[0167] The number of nucleus binding reagents in the nucleus isolation composition may vary in different implementations. In some embodiments, the number of nucleus binding reagents in the nucleus isolation composition 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, 8, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any number between two of these values The range may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of nuclei binding reagents in the nuclei isolation composition is 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, It may be 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0168] The nucleus binding reagent may bind to one or more components of the nucleus. In some embodiments, the number of nucleus binding reagents that bind to one component of the nucleus 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, 1 7, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any number between two of these values or range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of nucleus binding reagents that bind to one component of the nucleus is 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, 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, 111, 112, 113, 114, 115, 116, 11 It may be 3, 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0169] The number of nuclear components to which the nuclear binding reagent in the nuclear isolation composition can bind may vary in different implementations. In some embodiments, the number of nuclear components to which the nuclear binding reagent in the nuclear isolation composition can bind 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any two of these values 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, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of components of a nucleus to which a nucleus binding reagent in a nuclei isolation composition can bind is 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, 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, 111, 112, 113, 114, 115, It may be 1, 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0170] Method 400 may include barcoding multiple targets (e.g., mRNA molecules 416 shown in FIGS. 4A-4B) in multiple nuclei using multiple barcodes to generate multiple barcoded targets. Each of the multiple barcodes may include a molecular beacon sequence and a target binding region, and the molecular beacon sequences of at least two of the multiple barcodes may include different sequences. Method 400 may include obtaining sequencing data for the multiple barcoded targets. Method 400 may include estimating the number of each of the multiple targets in the multiple cells using the molecular beacon sequences of the multiple barcodes in the sequencing data. In FIGS. 4A-4B, cell 404 may include one or more mitochondria 424 having mitochondrial protein 428.

[0171] In some embodiments, isolating the plurality of nuclei includes contacting the plurality of nuclei from the plurality of cells with a nuclei isolation composition (e.g., nuclear envelope protein-specific antibodies 432a-432c shown in Figures 4A-4B) to generate nuclei bound to a nuclei-binding reagent. Isolating the plurality of nuclei may include isolating the nuclei bound to a nuclei-binding reagent using a reagent capable of specifically binding to the nuclei-binding reagent (e.g., epitope-binding reagent 444 shown in Figures 4A-4B).

[0172] Nuclear components and nuclear binding agents In some embodiments, the nucleus-binding reagent associates with a first epitope (e.g., epitope 436 shown in Figures 4A-4B), and the reagent capable of specifically binding to the nucleus-binding reagent comprises a first epitope-binding reagent. The first epitope may comprise biotin, a hapten, or a combination thereof. The hapten may comprise digoxigenin, 2,4-dinitrophenol, fluorescein, or a combination thereof. The reagent capable of specifically binding to the nucleus-binding reagent may comprise an anti-hapten antibody. The reagent capable of specifically binding to the nucleus-binding reagent may comprise avidin, streptavidin, neutravidin, or a combination thereof. In some embodiments, the first epitope and / or first epitope-binding reagent may be an affinity moiety such as biotin, streptavidin, heparin, an aptamer, a click chemistry moiety, digoxigenin, a primary amine, a carboxyl, a hydroxyl, an aldehyde, a ketone, or any combination thereof. The first epitope and first epitope-binding reagent may be members of a binding pair, e.g., biotin / streptavidin.

[0173] The number of different first epitopes associated with a nucleic binding reagent may vary in different implementations. In some embodiments, the number of different first epitopes associated with a nucleic binding reagent 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 or any two of these values or a number or range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or a number or range between any two of these values.In some embodiments, the number of different first epitopes associated with the nucleic binding reagent is 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, 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, 111, 112, 113, 114, 115, 116, 117, 118, It may be 6, 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 or 100.

[0174] The number of molecules of the first epitope associated with the nucleic binding reagent may vary in different implementations. In some embodiments, the number of molecules of the first epitope associated with the nucleic binding reagent 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 or any two of these values or a number or range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or a number or range between any two of these values.In some embodiments, the number of molecules of the first epitope associated with the nucleic binding reagent is 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, 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, 12 It may be 6, 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 or 100.

[0175] In some embodiments, the nucleus-binding reagent comprises a primary antibody capable of specifically binding to one or more components of the nucleus (e.g., nuclear envelope protein-specific antibodies 432a-432c shown in Figures 4A-4B), and the reagent capable of specifically binding to the nucleus-binding reagent comprises a secondary antibody capable of specifically binding to the primary antibody. In some embodiments, the nuclear binding reagent comprises a nuclear envelope surface component binding reagent (e.g., a nuclear envelope protein-specific antibody), which may be capable of specifically binding to one or more nuclear envelope surface components.

[0176] In some embodiments, the nucleic-binding reagent comprises a carbohydrate-binding reagent. The carbohydrate-binding reagent may comprise a carbohydrate-binding protein. The carbohydrate-binding protein may comprise a lectin. The lectin may comprise a mannose-binding lectin, a galactose-binding lectin, an N-acetylgalactosamine-binding lectin, an N-acetylglucosamine-binding lectin, an N-acetylneuraminic acid-binding lectin, a fucose-binding lectin, or a combination thereof. The lectin may include concanavalin A (ConA), lentil lectin (LCH), snowdrop lectin (GNA), castor bean (Ricinus communis) agglutinin (RCA), peanut agglutinin (PNA), jacalin (AIL), hairy vetch lectin (VVL), wheat germ agglutinin (WGA), elderberry lectin (SNA), Maackia amurensis leukoagglutinin (MAL), Maackia amurensis erythrocyte agglutinin (MAH), Ulex europaeus agglutinin (UEA), Aleuria aurantia lectin (AAL), or a combination thereof. The lectin may be or include an agglutinin. The agglutinin may be or include wheat germ agglutinin (WGA). The carbohydrate binding protein may be from or derived from an animal, a bacterium, a virus, a fungus, or a combination thereof. The carbohydrate binding protein may be from or derived from a plant.The plant may be jack bean (Canavalia ensiformis), lentil (Lens culinaris), snowdrop (Galanthus nivalis), castor bean (Ricinus communis), peanut (Arachis hypogaea), jackfruit (Artocarpus integrifolia), vicia villosa, wheat (Triticum vulgaris), elderberry (Sambucus nigra), maackia amurensis, gorse (Ulex europaeus), aleuria aurantia, or a combination thereof.

[0177] In some embodiments, one or more components of the core include a sugar, an oligosaccharide, a polysaccharide, a derivative thereof, or a combination thereof. The one or more components of the core may include a monosaccharide, a disaccharide, a polyol, a maltooligosaccharide, a non-maltooligosaccharide, a starch, a non-starch polysaccharide, a derivative thereof, or a combination thereof. The one or more components of the core may include glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, maltodextrin, raffinose, stachyose, a fructo-oligosaccharide, amylose, amylopectin, modified starch, glycogen, cellulose, hemicellulose, pectin, a hydrocolloid, a derivative thereof, or a combination thereof. One or more components of the core may be α-D-mannosyl residues, α-D-glucosyl residues, branched α-mannosidic structures of high α-mannose types, branched α-mannosidic structures of hybrid and biantennary complex N-glycans, fucosylated core regions of biantennary and triantennary complex N-glycans, α1-3- and α1-6-linked high mannose structures, Galβ1-4GalNAcβ1-R, Galβ1-3GalNAcα1-Ser / Thr, (Sia)Galβ1-3GalNAcα1-Ser / Thr, GalNAcα-Ser / Thr, GlcNAcβ1-4GlcNAcβ1-4GlcNAc, Neu5Ac (sialic acid), Neu5Acα2-6Gal(NAc)-R, Neu5Ac / The core may comprise Gcα2,3Galβ1,4Glc(NAc), Neu5Ac / Gcα2,3Galβ1,3(Neu5Acα2,6)GalNac, Fucα1-2Gal-R, Fucα1-2Galβ1-4(Fucα1-3 / 4)Galβ1-4GlcNAc, R2-GlcNAcβ1-4(Fucα1-6)GlcNAc-R1, derivatives thereof, or combinations thereof. One or more components of the core may comprise a glycoprotein, a glycolipid, or a combination thereof.

[0178] In some embodiments, the one or more components of the nucleus include lamin, emerin, nesprin, nurim, UNC-83, kral, ZYG-12, Kms1p, UNC-84, kraloid, SUN-1, Sad1p, LBR, MAN1, LAP1, LAP2, LINK, nuclear pore complex, portions thereof, or combinations thereof. The number of one or more components of the nucleus may vary in different implementations. In some embodiments, the number of one or more components of the core 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any number between two of these values may be in the range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of one or more components of the core is 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, It may be 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0179] nuclear isolation particles In some embodiments, the nucleus-binding reagent is associated with the nucleus isolation particle. A reagent capable of specifically binding to the nucleus-binding reagent may be associated with the nucleus isolation particle (particle 448 having a surface as shown in Figures 4A-4B). The reagent capable of specifically binding to the nucleus-binding reagent may be immobilized or partially immobilized on the nucleus isolation particle. For example, the reagent capable of specifically binding to the nucleus-binding reagent (e.g., epitope-binding reagent 444) may be reversibly, irreversibly, covalently, non-covalently, or a combination thereof associated with the nucleus isolation particle. As another example, the reagent capable of specifically binding to the nucleus-binding reagent may be embedded, partially embedded, not embedded, encapsulated, partially encapsulated, not encapsulated, or a combination thereof in the nucleus isolation particle. In some embodiments, the first epitope-binding reagent is associated with the nucleus isolation particle (e.g., immobilized, partially immobilized, encapsulated, or partially encapsulated) through a cleavable linker. A cleavable linker can operably link the first epitope-binding reagent to the nuclei isolation particle. The cleavable linker can include a chemically cleavable linkage, a photocleavable linkage, an acid-labile linker, a heat-sensitive linkage, an enzymatically cleavable linkage, or a combination thereof. Some embodiments of the methods and compositions disclosed herein provide null particles. In some embodiments, the null particles do not include multiple barcodes, magnetic properties, and / or a reagent (e.g., a first epitope-binding reagent) that can specifically bind to the nuclei-binding reagent. The null particles may resemble nuclei isolation particles in all aspects except for the absence of multiple barcodes, magnetic properties, and / or the first epitope-binding reagent. Some embodiments of the methods and compositions disclosed herein provide a liberated first epitope. The liberated first epitope can include all or a portion of the first epitope. In some embodiments, the liberated first epitope can bind to an unbound first epitope-binding reagent.

[0180] In some embodiments, the nucleus isolation particles comprise nucleus isolation beads. The nucleus isolation particles 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 combinations thereof. The nucleus isolation particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The nucleus isolation particles may be disintegrable. The nucleus isolation particles may comprise disintegrable nucleus isolation hydrogel particles.

[0181] In some embodiments, isolating the nuclei bound to the nuclei-binding reagent may include isolating the nuclei isolation particles by magnetic removal, centrifugation, or any combination thereof. In some embodiments, the plurality of barcodes are associated with the nuclei isolation particles. At least one barcode of the plurality of barcodes may be immobilized on the nuclei isolation particles. At least one barcode of the plurality of barcodes may be partially immobilized on the nuclei isolation particles. At least one barcode of the plurality of barcodes may be encapsulated within the nuclei isolation particles. At least one barcode of the plurality of barcodes may be partially encapsulated within the nuclei isolation particles. At least one barcode of the plurality of barcodes is not encapsulated within the nuclei isolation particles. At least one barcode of the plurality of barcodes may be embedded within the nuclei isolation particles. At least one barcode of the plurality of barcodes may be partially embedded within the nuclei isolation particles. At least one barcode of the plurality of barcodes is not embedded within the nuclei isolation particles.

[0182] Nuclear indexing oligonucleotides In some embodiments, the nucleus-binding reagent may include a nucleus-indexing oligonucleotide (e.g., barcode 440a associated with nuclear envelope protein-specific antibody 432a shown in Figures 4A-4B). The nucleus-indexing oligonucleotide may include a nucleus-indexing sequence. Method 400 may include barcoding the nucleus-indexing oligonucleotide using a plurality of barcodes (e.g., as a sample for indexing cell nuclei) to generate a plurality of barcoded nucleus-indexing oligonucleotides, and obtaining sequencing data for the plurality of barcoded nucleus-indexing oligonucleotides. Barcoding the nucleus-indexing oligonucleotide may include stochastically barcoding the nucleus-indexing oligonucleotide using a plurality of barcodes to generate a plurality of barcoded nucleus-indexing oligonucleotides. Barcoding the nucleus-indexing oligonucleotide using a plurality of barcodes may include contacting the plurality of barcodes with the nucleus-indexing oligonucleotide to generate barcodes hybridized to the nucleus-indexing oligonucleotides, and extending the barcodes hybridized to the nucleus-indexing oligonucleotide to generate a plurality of barcoded nucleus-indexing oligonucleotides. Extending the barcode may include extending the barcode using a DNA polymerase to generate a plurality of barcoded nuclear-indexing oligonucleotides. Extending the barcode may include extending the barcode using a reverse transcriptase to generate a plurality of barcoded nuclear-indexing oligonucleotides.

[0183] The number of nucleus-indexing oligonucleotides associated with (e.g., bound to, conjugated to, etc.) a nucleus-binding reagent may vary in different implementations. In some embodiments, the number of nucleus-indexing oligonucleotides associated with a nucleus-binding reagent 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any two of these values 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, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of nucleus indexing oligonucleotides associated with a nucleus binding reagent is 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, 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0184] The nuclear indexing oligonucleotides associated with the nuclear binding reagents may have the same sequence (or the same nuclear indexing sequence) or different sequences (or different nuclear indexing sequences). In some embodiments, the number of nuclear indexing oligonucleotides associated with the nuclear binding reagents having the same sequence (or the same nuclear indexing 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, 200, 300, 400, 500, 600, 700, 800, 900, 100 or and the like. The present invention may be a number or range between any two of these values, or may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 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, 200, 300, 400, 500, 600, 700, 800, 900, 100 or a number or range between any two of these values.In some embodiments, the number of nucleus indexing oligonucleotides associated with a nucleus binding reagent having the same sequence (or the same nucleus indexing sequence) is 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, It may be 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.In some embodiments, the number of nucleus indexing oligonucleotides associated with nucleus binding reagents having different sequences (or different nucleus indexing sequences) 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or and the like. The present invention may be a number or range between any two of these values, or may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of nuclear indexing oligonucleotides associated with a nuclear binding reagent having different sequences (or different nuclear indexing sequences) is 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, It may be 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0185] In some embodiments, method 400 may include amplifying a plurality of barcoded nuclear indexing oligonucleotides to generate a plurality of barcoded nuclear indexing amplicons. Amplifying a plurality of barcoded nuclear indexing oligonucleotides may include amplifying at least a portion of the molecular label sequences and at least a portion of the nuclear indexing oligonucleotides using polymerase chain reaction (PCR). Obtaining sequencing data of the plurality of barcoded nuclear indexing oligonucleotides may include obtaining sequencing data of the plurality of barcoded nuclear indexing amplicons. Obtaining sequencing data of the plurality of barcoded nuclear indexing oligonucleotides may include sequencing at least a portion of the molecular label sequences and at least a portion of the nuclear indexing oligonucleotides.

[0186] Barcoded particles In some embodiments, the plurality of barcodes are associated with a barcoded particle. At least one barcode of the plurality of barcodes may be immobilized on the barcoded particle. At least one barcode of the plurality of barcodes may be partially immobilized on the barcoded particle. At least one barcode of the plurality of barcodes may be encapsulated within the barcoded particle. At least one barcode of the plurality of barcodes may be partially encapsulated within the barcoded particle. The barcoded particle may be disintegrable. The barcoded particle may comprise a barcoded bead. The barcoded particle may comprise a sepharose bead, a streptavidin bead, an agarose bead, a magnetic bead, a conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein A / G conjugated bead, a protein L conjugated bead, an oligo(dT) conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, an anti-fluorescent dye microbead, or a combination thereof. The barcoded particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The barcoded particles may comprise disintegrable hydrogel particles.

[0187] Barcodes and barcoding In some embodiments, barcoding the multiple targets using the multiple barcodes to generate the multiple barcoded targets includes contacting copies of the targets with target binding regions of the barcodes and reverse transcribing the multiple targets using the multiple barcodes to generate the multiple barcoded targets. Method 400 may include amplifying the multiple barcoded targets to generate the multiple amplified barcoded targets prior to obtaining sequencing data for the multiple barcoded targets. Amplifying the barcoded targets to generate the multiple amplified barcoded targets may include using polymerase chain reaction (PCR) to amplify the barcoded targets to generate the multiple amplified barcoded targets. Amplifying the multiple amplified barcoded targets may include amplifying a molecular beacon sequence and a sequence of one of the multiple targets, or a portion thereof, to generate the multiple barcoded target amplicons. Amplifying the plurality of amplified barcoded targets may include amplifying the plurality of amplified barcoded targets using polymerase chain reaction (PCR) to generate amplicons of the plurality of barcoded targets. Barcoding the plurality of cellular targets using the plurality of barcodes to generate the plurality of barcoded targets may include stochastically barcoding the plurality of cellular targets using a plurality of stochastic barcodes to generate the plurality of stochastically barcoded targets.

[0188] In some embodiments, each of the plurality of barcodes comprises a cell label sequence, a binding site for a universal primer, or any combination thereof, and the cell label sequences of at least two barcodes of the plurality of barcodes comprise the same sequence. The target binding region may comprise a poly(dT) region. At least 100 molecular label sequences of the plurality of barcodes may comprise different sequences. At least 1000 molecular label sequences of the plurality of barcodes may comprise different sequences. At least 10,000 molecular label sequences of the plurality of barcodes may comprise different sequences. The molecular label sequences of the plurality of barcodes may comprise random sequences. The target binding region may comprise a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof.

[0189] Cell lysis In some embodiments, method 400 includes lysing the plurality of nuclei before barcoding the plurality of targets in the plurality of nuclei using the plurality of barcodes to generate the plurality of barcoded targets. In some embodiments, method 400 includes lysing the plurality of cells without lysing the nuclei of the plurality of cells before isolating the plurality of nuclei of the plurality of cells using a nuclei isolation composition.

[0190] Nuclear Barcoding Compositions The present disclosure includes embodiments of barcoded compositions. In some embodiments, the barcoded composition includes a nuclei isolation composition comprising a nuclei-binding reagent, wherein the nuclei-binding reagent can specifically bind to one or more components of a nucleus; and a plurality of barcodes, each of the plurality of barcodes comprising a molecular beacon sequence and a target binding region, wherein the molecular beacon sequences of at least two of the plurality of barcodes comprise different sequences. In some embodiments, the composition includes a reagent capable of specifically binding to the nuclei-binding reagent. The nuclei-binding reagent may be associated with a first epitope, and the reagent capable of specifically binding to the nuclei-binding reagent may comprise a first epitope-binding reagent. In some embodiments, the first epitope-binding reagent is associated with the nuclei isolation particle (e.g., immobilized, partially immobilized, encapsulated, partially encapsulated) through a cleavable linker. The cleavable linker may operably link the first epitope-binding reagent to the nuclei isolation particle. The cleavable linker may comprise a chemically cleavable linkage, a photocleavable linkage, an acid-labile linker, a heat-sensitive linkage, an enzymatically cleavable linkage, or a combination thereof. In some embodiments, the nuclei-binding reagent comprises a primary antibody capable of specifically binding to one or more components of a nucleus, and the reagent capable of specifically binding to the nuclei-binding reagent comprises a secondary antibody capable of specifically binding to the primary antibody. In some such embodiments, the secondary antibody is linked to the nuclei-isolation particle through a cleavable linker. In some embodiments, the nuclei-binding reagent comprises a carbohydrate-binding reagent. The carbohydrate-binding reagent may comprise a carbohydrate-binding protein. In some embodiments, the nuclei-binding reagent is associated with the nuclei-isolation particle. In some embodiments, a plurality of barcodes is associated with the nuclei-isolation particle. In some embodiments, the nuclei-binding reagent may comprise a nuclei-indexing oligonucleotide, and the nuclei-indexing oligonucleotide comprises a nuclei-indexing sequence. A plurality of barcodes may be associated with the barcoded particle.

[0191] Organelle removal methods and compositions The disclosure herein also includes methods, compositions, and systems for performing organelle removal or isolation for single-cell transcriptome analysis (or other single-cell omics or multiomics analysis, e.g., proteomics analysis to determine the protein expression profile of a single cell). In some embodiments, the organelle removal step can be performed using antibodies, e.g., antibodies associated with epitopes. For example, antibodies conjugated to one or more strong epitopes can be used for the organelle removal step. The organelle removal step can provide less contaminating cytoplasmic and / or nuclear RNA for NGS library preparation. For example, purified or cleaner cytoplasmic and / or nuclear RNA can be obtained for NGS library preparation. The organelle removal step can be useful for single-cell library preparation because organelle-specific RNA, e.g., mitochondrial RNA, can contribute to sequencing reads (e.g., the majority of sequencing reads). In some embodiments, the organelle removal step includes removal of mitochondria with a mitochondria-specific antibody (or another mitochondria-specific binding reagent). For example, the organelle removal step may involve removing mitochondria with a mitochondrial-specific antibody (or another mitochondrial-specific binding reagent) in a single step while performing one or more downstream processes on the mitochondrial-depleted pool of RNA. Downstream processes may include cDNA synthesis for transcriptome analysis and nucleic acid extension reactions for single-cell proteomics analysis. In some embodiments, mitochondrial contaminants may be reduced (e.g., eliminated) in single-cell analysis (e.g., whole transcriptome analysis or proteomics analysis).

[0192] In some embodiments, barcoding and isolation of nuclei can occur in a single step. In some embodiments, depletion of mitochondrial content (e.g., RNA) can occur at the beginning of library preparation. In some embodiments, barcoding and isolation of nuclei and organelle removal can occur in a single step or can be completed in time.

[0193] 5A-5B show non-limiting, exemplary schematic diagrams of a barcoding workflow (e.g., stochastic barcoding) involving organelle (e.g., mitochondrial) removal. Method 500 can include cell lysis (e.g., using a light lysis buffer that lyses cells without lysing one or more organelles), organelle binding and removal (e.g., mitochondria binding and removal), and subsequent single-cell analysis (e.g., single-cell expression profile analysis). For example, selective removal of organelles such as mitochondria with antibodies associated with (e.g., conjugated to) epitopes (e.g., strong epitopes such as biotin, fluorescein, and DIG) can reduce (e.g., minimize or avoid) contaminants in NGS libraries. The organelle removal step can be part of a single-cell workflow, such as nuclear barcoding method 400 described with reference to FIGS. 4A-4B or the Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ).

[0194] In some embodiments, method 500 includes, prior to or during isolating a plurality of nuclei of a plurality of cells using a nuclei isolation composition, penetrating the plurality of cells (e.g., selectively permeabilizing and / or lysing the plasma membrane while preserving the membranes of one or more organelles intact), and depleting one or more organelles (e.g., mitochondria 524) of the plurality of cells using an organelle capture composition (e.g., antibody 532a shown in Figures 5A-5B) comprising an organelle-binding reagent, where the organelle-binding reagent can specifically bind to one or more components of the one or more organelles of the plurality of cells. Depleting the one or more organelles may include contacting one or more organelles of the plurality of cells with the organelle capture composition to generate one or more organelles bound to the organelle component-binding reagent. Depleting the one or more organelles may include depleting the one or more organelles bound to the organelle-binding reagent using a reagent capable of specifically binding to the organelle-binding reagent. The organelle-binding reagent may be associated with a second epitope (e.g., epitope 536), and the reagent capable of specifically binding to the organelle-binding reagent (e.g., epitope-binding reagent 544) may include the second epitope-binding reagent.

[0195] The number of organelle binding reagents in an organelle capture composition may vary in different implementations. In some embodiments, the number of organelle binding reagents in an organelle capture composition 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 6, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any two values ​​between these or may be a number or range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 2 It may be 1, 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of organelle binding reagents in an organelle capture composition is 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, 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0196] The number of organelle-binding reagents in an organelle-capturing composition that bind to an organelle may vary in different implementations. In some embodiments, the number of organelle-binding reagents in an organelle-capturing composition that bind to an organelle 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any of these values or a number or range between two thereof, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of organelle binding reagents in an organelle capture composition that bind to an organelle is 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, It may be 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0197] The number of organelles to which an organelle-binding reagent in an organelle-capturing composition can bind may vary in different implementations. In some embodiments, the number of organelles to which an organelle-binding reagent in an organelle-capturing composition can bind 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any of these values or a number or range between two thereof, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of organelles to which the organelle-binding reagent in the organelle capture composition can bind is 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, It may be 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0198] The number of different organelles removed or depleted may vary in different implementations. In some embodiments, the number of different organelles removed or depleted 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any number between two of these values or range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of different organelles removed or depleted is 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, It may be 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0199] The number of molecules of an organelle removed or depleted may vary in different embodiments. In some embodiments, the number of molecules of an organelle removed or depleted 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, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or any number between two of these values or range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or a number or range between any two of these values.In some embodiments, the number of molecules of an organelle removed or depleted is 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, It may be 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, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.

[0200] In some embodiments, the second epitope may comprise biotin, a hapten, or a combination thereof. The hapten may comprise digoxigenin, 2,4-dinitrophenol, fluorescein, or a combination thereof. The reagent capable of specifically binding to the organelle-binding reagent may comprise an anti-hapten antibody. The reagent capable of specifically binding to the organelle-binding reagent may comprise avidin, streptavidin, neutravidin, or a combination thereof. In some embodiments, the second epitope and / or second epitope-binding reagent may be an affinity moiety such as biotin, streptavidin, heparin, an aptamer, a click chemistry moiety, digoxigenin, a primary amine, a carboxyl, a hydroxyl, an aldehyde, a ketone, or any combination thereof. The second epitope and second epitope-binding reagent may be members of a binding pair, e.g., biotin / streptavidin. The organelle-binding reagent may comprise a primary antibody capable of specifically binding to one or more components of one or more organelles of the plurality of cells, and the reagent capable of specifically binding to the organelle-binding reagent may comprise a secondary antibody capable of specifically binding to the primary antibody.

[0201] In some embodiments, the organelle binding reagent may comprise an organelle surface component binding reagent, wherein one or more components of one or more organelles may comprise one or more organelle surface components, and the organelle binding reagent may be capable of specifically binding to one or more organelle surface components.

[0202] Organelle-capturing particles In some embodiments, a reagent capable of specifically binding to an organelle-binding reagent (e.g., a second epitope-binding reagent) is associated with an organelle-capturing particle (e.g., particle 548 having a surface as shown in Figures 5A-5B). The reagent capable of specifically binding to an organelle-binding reagent may be immobilized or partially immobilized on the organelle-capturing particle. For example, the reagent capable of specifically binding to an organelle-binding reagent may be reversibly, irreversibly, covalently, noncovalently, or a combination thereof associated with the organelle-capturing particle. As another example, the reagent capable of specifically binding to an organelle-binding reagent may be embedded, partially embedded, non-embedded, encapsulated, partially encapsulated, non-encapsulated, or a combination thereof in the organelle-capturing particle.

[0203] In some embodiments, the organelle capture particle comprises an organelle capture bead. The organelle capture particle may comprise Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugate beads, protein A conjugate beads, protein G conjugate beads, protein A / G conjugate beads, protein L conjugate beads, oligo(dT) conjugate beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The organelle capture particle may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof.

[0204] In some embodiments, depleting organelles from the plurality of cells using the organelle-capturing composition can include depleting one or more organelle-capturing particles by magnetic removal, centrifugation, or any combination thereof. The organelles can include mitochondria from the plurality of cells. One or more components of one or more organelles from the plurality of cells can include ABCD3, ESR2, NOS3, ALB, HIF1A, NR3C1, ATP5A1, HK1, PGR, CASQ1, HSPA1A, PHB, CLTC, HSPD1, PLN, COX4I1, IFM1, SOD1, CPS1, LGALS3, TP53, cytochrome C oxidase, MAPT, TP5B, ERN1, MT-CO1, VDAC1, or a combination thereof.

[0205] Organelle-removing composition In some embodiments, the composition comprises an organelle capture composition comprising an organelle-binding reagent, wherein the organelle-binding reagent is capable of specifically binding to one or more components of one or more organelles. The composition may comprise a reagent capable of specifically binding to the organelle-binding reagent. The organelle-binding reagent may be associated with a second epitope, and the reagent capable of specifically binding to the organelle-binding reagent may comprise a second epitope-binding reagent. In some embodiments, the reagent capable of specifically binding to the organelle-binding reagent is associated with an organelle capture particle. In some embodiments, the organelle-binding reagent comprises an organelle surface component-binding reagent, and the one or more components of the one or more organelles may comprise one or more organelle surface components, and the organelle-binding reagent may be capable of specifically binding to one or more organelle surface components.

[0206] Sequencing In some embodiments, estimating the number of distinct barcoded targets (e.g., stochastically barcoded targets) may include determining the sequence of the labeled targets, spatial labels, molecular labels, sample labels, cellular labels, or any products thereof (e.g., labeled amplicons, or labeled cDNA molecules). The amplified targets may be subjected to sequencing. Determining the sequence of the barcoded targets (e.g., stochastically barcoded targets) or any products thereof may include performing a sequencing reaction to determine the sequence of at least a portion of the sample labels, spatial labels, cellular labels, molecular labels, at least a portion of the labeled targets (e.g., stochastically labeled targets), their complements, their reverse complements, or any combination thereof.

[0207] Determining the sequence of a barcoded target or a stochastically barcoded target (e.g., amplified nucleic acid, labeled nucleic acid, cDNA copy of labeled nucleic acid, etc.) can be accomplished using techniques such as, but not limited to, sequencing by hybridization (SBH), sequencing by ligation (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan reporter probe digestion, pyrosequencing, fluorescence in This can be performed using a variety of sequencing methods, including in situ sequencing (FISSEQ), FISSEQ beads, wobble sequencing, multiplex sequencing, polymerized colony (POLONY) sequencing; nanogrid rolling circle sequencing (ROLONY), allele-specific oligo ligation assays (e.g., oligo ligation assay (OLA), single template molecule OLA using ligated linear probes and rolling circle amplification (RCA) readout, ligated padlock probes, or single template molecule OLA using ligated circular padlock probes and rolling circle amplification (RCA) readout), etc.

[0208] In some embodiments, determining the sequence of the barcoded target (e.g., a stochastically barcoded target) or any product thereof comprises paired-end sequencing, nanopore sequencing, high-throughput sequencing, shotgun sequencing, dye-terminator sequencing, multiple-primer DNA sequencing, primer walking, Sanger dideoxy sequencing, Maxim Gilbert sequencing, pyrosequencing, true single-molecule sequencing, or any combination thereof. Alternatively, the sequence of the barcoded target or any product thereof can be determined by electron microscopy or chemical-sensitive field-effect transistor (chemFET) arrays. High-throughput sequencing methods such as circular array sequencing using platforms such as Roche 454, Illumina Solexa, ABI-SOLiD, ION Torrent, Complete Genomics, PacificBioscience, Helicos, or Polonator platforms can also be utilized. In some embodiments, sequencing can include MiSeq sequencing. In some embodiments, sequencing can include HiSeq sequencing.

[0209] The labeled targets (probabilistically labeled targets) may comprise nucleic acids representing between about 0.01% and about 100% of the genes in an organism's genome. For example, by using a target-complementary region comprising multiple multimers to capture genes containing complementary sequences in a sample, it is possible to sequence between about 0.01% and about 100% of the genes in an organism's genome. In some embodiments, the barcoded targets comprise nucleic acids representing between about 0.01% and about 100% of the transcripts in an organism's transcriptome. For example, by using a target-complementary region comprising a poly(T) tail to capture mRNA from a sample, it is possible to sequence between about 0.501% and about 100% of the transcripts in an organism's transcriptome.

[0210] Determining the sequence of spatial and molecular labels of the plurality of barcodes (e.g., stochastic barcodes) may include sequencing 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, 100%, or a number or range between any two of these values ​​of the plurality of barcodes. Determining the sequence of labels, e.g., sample labels, spatial labels, and molecular labels, of the plurality of barcodes may include sequencing 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100% of the plurality of barcodes. 3 , 104 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , 10 20 Sequencing some or all of the plurality of barcodes may include generating sequences of read lengths of, about, at least, or at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or a number or range between any two of these values.

[0211] The sequencing step may include sequencing at least, or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides or base pairs of the barcoded targets. For example, the sequencing step may include performing polymerase chain reaction (PCR) amplification on a plurality of barcoded targets to generate sequencing data having sequences with read lengths of 50, 75, or 100 or more nucleotides. The sequencing step may include sequencing at least, or at least about 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more nucleotides or base pairs of the barcoded targets. The sequencing step may include sequencing at least, or at least about 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 or more nucleotides or base pairs of the barcoded target.

[0212] The sequencing step may include at least about 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more sequencing reads per run. In some embodiments, the sequencing step includes at least, or at least about 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, or 10,000, or more sequencing reads per run. The sequencing step may include no more than about 1,600,000,000 sequencing reads per run. The sequencing step may include no more than about 200,000,000 reads per run.

[0213] sample In some embodiments, multiple targets may be included in one or more samples. A sample may include one or more cells or nucleic acids from one or more cells. A sample may be a single cell or nucleic acids from a single cell. The one or more cells may be of one or more cell types. At least one of the one or more cell types may be a brain cell, a cardiac cell, a cancer cell, a circulating tumor cell, an organ cell, an epithelial cell, a metastatic cell, a benign cell, a primary cell, a circulating cell, or any combination thereof.

[0214] A sample for use in the methods of the present disclosure may contain one or more cells. A sample may refer to one or more cells. In some embodiments, a plurality of cells may include one or more cell types. At least one of the one or more cell types may be brain cells, cardiac cells, cancer cells, circulating tumor cells, organ cells, epithelial cells, metastatic cells, benign cells, primary cells, circulating cells, or any combination thereof. In some embodiments, the cells are cancer cells excised from cancer tissue, such as breast cancer, lung cancer, colon cancer, prostate cancer, ovarian cancer, pancreatic cancer, brain cancer, melanoma, and non-melanoma skin cancer. In some embodiments, the cells are derived from cancer but collected from bodily fluids (e.g., circulating tumor cells). Non-limiting examples of cancers include adenoma, adenocarcinoma, squamous cell carcinoma, basal cell carcinoma, small cell carcinoma, large cell undifferentiated carcinoma, chondrosarcoma, and fibrosarcoma. A sample may include tissue, cell monolayers, fixed cells, tissue sections, or any combination thereof. The sample may include a biological sample, a clinical sample, an environmental sample, a biological fluid, a tissue, or cells from a subject. The sample may be obtained from a human, a mammal, a dog, a rat, a mouse, a fish, a fly, a worm, a plant, a fungus, a bacterium, a virus, a vertebrate, or an invertebrate.

[0215] In some embodiments, the cell is a cell infected with a virus and containing viral oligonucleotides. In some embodiments, the viral infection may be caused by a virus such as a single-stranded (positive or "sense") DNA virus (e.g., parvovirus) or a double-stranded RNA virus (e.g., retrovirus). In some embodiments, the cell is a bacterium. These may include either gram-positive or gram-negative bacteria. In some embodiments, the cell is a fungus. In some embodiments, the cell is a protozoan or other parasite.

[0216] As used herein, the term "cell" can refer to one or more cells. In some embodiments, the cells are normal cells, e.g., human cells at different developmental stages, or human cells derived from different organs or tissue types. In some embodiments, the cells are non-human cells, e.g., other types of mammalian cells (e.g., mouse, rat, pig, dog, cow, or horse). In some embodiments, the cells are other types of animal or plant cells. In other embodiments, the cells may be any prokaryotic or eukaryotic cell.

[0217] In some embodiments, the cells are sorted before associating the cells with the beads. For example, the cells can be sorted by fluorescence-activated cell sorting or magnetic-activated cell sorting, or more commonly by flow cytometry. The cells can also be filtered by size. In some embodiments, the retentate contains the cells associated with the beads. In some embodiments, the flow-through contains the cells associated with the beads. A sample may refer to a plurality of cells. A sample may refer to a monolayer of cells. A sample may refer to a thin section (e.g., a tissue slice). A sample may refer to a solid or semi-solid collection of cells that can be arranged in a one-dimensional array.

[0218] Methods for removing undesired nucleic acid species Single-cell analysis, such as single-cell whole transcriptome analysis (WTA), can be hindered by the presence or high abundance of unintended targets or target species (e.g., unintended nucleic acids or nucleic acid species, also referred to herein as unwanted or unnecessary nucleic acids or nucleic acid species). In many samples, nucleic acids derived from non-nuclear organelles (e.g., mitochondrial mRNA and ribosomal RNA) can be highly abundant and / or undesirable. For example, for heterogeneous cDNA samples, PCR is typically performed with excessive cycles to adequately amplify low-expressor genes; in these scenarios, native gene expression profiles are typically skewed by dominant high-expressor PCR products. A method to correct for this bias in PCR products is molecular indexing; however, because high-expressor genes, such as mitochondrial mRNA, often dominate sequencing runs while contributing little to experimental interpretation, the sequencing cost relative to molecular index counts is considered high. Previous efforts to increase the relative abundance of low-abundance species in nucleic acid samples have included library normalization methods. Some embodiments disclosed herein provide methods for removing undesired nucleic acid species (eg, nucleic acids derived from non-nuclear organelles, mtRNA, rRNA) from a plurality of nucleic acid molecules.

[0219] In some embodiments, the methods disclosed herein include providing a sample containing a plurality of nucleic acid target molecules. The sample may, in some embodiments, contain one or more undesired nucleic acid species. It will be recognized by those skilled in the art that the plurality of nucleic acid target molecules and / or undesired nucleic acid species may include a variety of nucleic acid target molecules. For example, the nucleic acid target molecules and / or undesired nucleic acid species may include DNA molecules, RNA molecules, genomic DNA molecules, cDNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or any combination thereof. The nucleic acid target molecules may be double-stranded or single-stranded. In some embodiments, the plurality of nucleic acid target molecules may include nucleic acids derived from one or more non-nuclear organelles. In some embodiments, the plurality of nucleic acid target molecules may include polyA RNA molecules. In some embodiments, the plurality of nucleic acid target molecules comprises at least 100, at least 1,000, at least 10,000, at least 20,000, at least 30,000, at least 40,000, at least 50,000, at least 100,000, at least 1,000,000, or more nucleic acid species.

[0220] A sample may contain, for example, multiple nucleic acid target molecules and one or more undesired nucleic acid species (e.g., nucleic acids derived from non-nuclear organelles). The one or more undesired nucleic acid species may be present in a sample in different amounts. For example, the one or more undesired nucleic acid species may represent about or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or a higher percentage, or 100%, of the nucleic acid content of the sample, or a range between two of these values. In some embodiments, the one or more undesired nucleic acid species represent at least about 10% of the nucleic acid content of the sample. In some embodiments, the one or more undesired nucleic acid species represent at least about 30% of the nucleic acid content of the sample. In some embodiments, the one or more undesired nucleic acid species account for at least about 50% of the nucleic acid content of the sample. The undesired nucleic acid species may be or include various types of nucleic acid molecules. For example, at least one of the one or more undesired nucleic acid species may be a ribosomal protein mRNA, a mitochondrial mRNA, a genomic DNA, an intron sequence, a highly abundant sequence, or a combination thereof. In some embodiments, the undesired nucleic acid species is or includes an mRNA species. The mRNA species may be, for example, one or more of: a housekeeping gene mRNA species, a ribosomal gene mRNA species, a mitochondrial gene mRNA species, a highly abundant gene mRNA species, or any combination thereof. In some embodiments, the undesired nucleic acid species is or includes a DNA species. The DNA species may be, for example, one or more of: a housekeeping gene DNA species, a ribosomal gene DNA species, a mitochondrial gene DNA species, a highly abundant gene DNA species, or any combination thereof. In some embodiments, the undesired nucleic acid species is or includes a DNA species and an RNA species.

[0221] In some embodiments, the nuclear isolation and / or organelle depletion methods described herein can significantly reduce the abundance of one or more undesired nucleic acid species (e.g., nucleic acids derived from non-nuclear organelles) compared to multiple nucleic acid target molecules in a sample. In some embodiments, the methods and compositions disclosed herein can reduce the abundance of one or more undesired nucleic acid species in a sample. For example, the methods and compositions disclosed herein can reduce the abundance of at least one, at least two, at least three, at least four, at least five, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000, or more undesired nucleic acid species in a sample. In some embodiments, the methods and compositions disclosed herein may reduce the abundance of one or more undesired nucleic acid species in a sample by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100%. In some embodiments, the abundance of one or more undesired nucleic acid species may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 55%, at least 50%, at least 65%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or more, or a range between any two of these values, compared to the abundance of at least one of the nucleic acid target molecules in the starting sample prior to organelle depletion and / or nuclear isolation described herein.

[0222] It will be appreciated that in some embodiments, the methods and compositions disclosed herein can reduce the abundance of undesired nucleic acid species without significantly reducing the number of different molecular label sequences associated with other nucleic acid target molecules.For example, the methods and compositions disclosed herein can reduce the abundance of undesired nucleic acid species while retaining at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the different molecular label sequences associated with other nucleic acid target molecules. In some embodiments, the methods and compositions disclosed herein can reduce the abundance of undesired nucleic acid species by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, while retaining at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the different molecular label sequences associated with other nucleic acid target molecules. In some embodiments, reducing the abundance of undesired nucleic acid species does not significantly reduce the number of different molecular label sequences associated with other nucleic acid target molecules.

[0223] In some embodiments, after using the nucleus isolation and / or organelle depletion methods described herein, the sequencing reads related to undesired nucleic acid species are less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or a lower percentage of the total sequencing reads. In some embodiments, the sequencing reads related to undesired nucleic acid species are less than 40% of the total sequencing reads. In some embodiments, the sequencing reads related to undesired nucleic acid species are less than 30% of the total sequencing reads. In some embodiments, the sequencing reads related to undesired nucleic acid species are less than 20% of the total sequencing reads. In some embodiments, the sequencing reads related to undesired nucleic acid species are less than 10% of the total sequencing reads. In some embodiments, after using the nuclei isolation and / or organelle depletion methods described herein, the sequencing reads for undesired nucleic acid species are reduced to less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the sequencing reads for undesired nucleic acids without using the nuclei isolation and / or organelle depletion methods described herein. In some embodiments, after using the nuclear isolation and / or organelle depletion methods described herein, the sequencing reads for undesired nucleic acid species are reduced to 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or a range between any two of these values, of the sequencing reads for undesired nucleic acids when not using the nuclear isolation and / or organelle depletion methods described herein.

[0224] In some embodiments, the methods and compositions disclosed herein can improve sequencing efficiency by reducing the ratio of sequencing reads:molecular labels of undesired nucleic acid species and / or increasing the ratio of sequencing reads:molecular labels of nucleic acid target molecules.For example, the ratio of sequencing reads to molecular labels of undesired nucleic acid species can be less than 20, less than 15, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1.In some embodiments, the ratio of sequencing reads to molecular labels of undesired nucleic acid species is 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or a range between any two of these values. [Example]

[0225] 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.

[0226] Example 1 Nuclei capture and barcoding workflow This example demonstrates a workflow for nuclei capture and barcoding. Figures 6A-6F are schematic diagrams of non-limiting exemplary workflows for nuclei isolation and single-cell profiling. The workflow may include cell lysis (e.g., using a photolysis buffer that dissolves the plasma membrane without lysing the nuclear membrane and / or one or more organelles). The workflow may include selective depletion of one or more organelles (e.g., mitochondria). The workflow may include associating each nucleus with a nuclei-indexing oligonucleotide containing a nuclei-indexing sequence (e.g., for sample indexing). The workflow may include isolating nuclei from one or more cellular components. The workflow may include dividing individual nuclei into compartments and performing nuclei lysis. The workflow may include a step of barcoding (e.g., stochastically barcoding) target nucleic acid molecules and / or nuclear indexing oligonucleotides for single cell analysis (e.g., Rhapsody™ Assay (Becton, Dickinson and Company, Franklin Lakes, NJ), Chromium™ Single Cell 3' Solution (10X Genomics, San Francisco, CA)).

[0227] Sample Composition A sample 601 may contain a plurality of cells 602. Samples for use in the disclosed methods may be those from which it is technically difficult or impossible to generate a single-cell suspension (e.g., frozen cells, fixed cells, tissue specimens, tumor specimens, epithelial tissue, formalin-fixed paraffin-embedded cells, and combinations thereof). The cells may include a plasma membrane 604 and a cell nucleus 606. The cell nucleus may include a nuclear envelope 608 and one or more nucleic acid target molecules 612. The nuclear envelope may include one or more nuclear envelope surface components 610, such as nuclear envelope proteins (e.g., lamins). The nucleus may include one or more target nucleic acid molecules (e.g., messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (long ncRNA or lncRNA), Piwi-interacting RNA (piRNA)). A cell may include one or more organelles described herein (e.g., ribosomes, mitochondria), and one or more organelles may include one or more organelle surface components. Mitochondria 616 may include organelle surface components (e.g., mitochondrial surface components 618). A cell may also include extranuclear cellular components 614. Extranuclear cellular components may include, for example, unwanted extranuclear cytoplasmic components (e.g., molecules that are unnecessary and / or deleterious to single-cell expression profiling). Extranuclear cellular components may include cytoplasmic fragments. Extranuclear cellular components may include organelles. Extranuclear cellular components may include mitochondria. Extranuclear cellular components may include ribosomes. Extranuclear cellular components may include rough endoplasmic reticulum. Extranuclear cellular components may include one or more unwanted nucleic acids (e.g., ribosomal RNA, mitochondrial DNA, mitochondrial RNA). Extranuclear cellular components may include one or more macromolecules that interfere with the single-cell expression analysis methods disclosed herein.The extranuclear cellular components 614 can include, for example, mitochondria, peroxisomes, cytosol, vesicles, lysosomes, plasma membrane, chloroplasts, intramitochondrial matrix, inner mitochondrial membrane, intermembrane space, outer mitochondrial membrane, secretory vesicles, smooth endoplasmic reticulum, rough endoplasmic reticulum, Golgi apparatus, phagosomes, endosomes, exosomes, plasma membrane, microtubules, microfilaments, intermediate filaments, filopodia, ruffles, lamellipodia, sarcomeres, focal adhesions, podosomes, ribosomes, microsomes, lipid rafts, cell wall, and combinations thereof.

[0228] Plasma membrane lysis The workflow may include plasma membrane lysis (step 600a) of a population of cells 602 of a sample 601. Plasma membrane lysis may produce a lysate containing multiple populations of nuclei, organelles, and extranuclear cellular components (e.g., ribosomes), as shown in FIG. 6A. The method may include lysing multiple cells without lysing the nuclear envelope. The method may include lysing multiple cells without lysing organelle membranes. The method may include lysing multiple cells and lysing organelle membranes without lysing the nuclear envelope. Plasma membrane lysis may be performed using a homogenizer (e.g., a Dounce homogenizer), detergent, or enzymatic methods.

[0229] Contacting with an organelle surface component binding reagent The workflow may include contacting the lysate with one or more organelle surface component-binding reagents (step 600b). A second epitope 622 may be associated (e.g., immobilized, partially immobilized, encapsulated, or partially encapsulated) with the organelle surface component-binding reagent 620 (e.g., the organelle-binding reagent). The second epitope 622 may include a strong epitope (e.g., biotin, fluorescein, and / or DIG) described herein. The organelle surface component-binding reagent 620 may be capable of specifically binding to one or more components of one or more organelles (e.g., mitochondria), such as mitochondrial surface component 618 (as shown in FIG. 6B). Contacting one or more organelles of the plurality of cells with the organelle surface component-binding reagent may result in one or more organelles bound to the organelle component-binding reagent. The organelle surface component binding reagent 620 may comprise a mitochondria-specific antibody (or another mitochondria-specific binding reagent).

[0230] Contacting with organelle-capturing particles The workflow may include contacting the lysate with one or more organelle capture particles (step 600c). The organelle capture particles 630 may include a reagent capable of specifically binding to an organelle-binding reagent described herein (e.g., the second epitope-binding reagent 632 shown in FIG. 6B). The second epitope-binding reagent 632 may be associated with (e.g., immobilized, partially immobilized, encapsulated, or partially encapsulated by) the organelle capture particles 630. In some embodiments, the organelle surface component-binding reagent 620 does not include a second epitope. In some such embodiments, the organelle surface component-binding reagent may include a primary antibody capable of specifically binding to one or more components of one or more organelles (e.g., mitochondria), and the reagent capable of specifically binding to the organelle-binding reagent includes a secondary antibody capable of specifically binding to the primary antibody. Step 600c may result in one or more organelles bound to the organelle capture particles via the organelle surface component-binding reagent.

[0231] Removal of organelle-trapped particles The workflow may include removing the organelle-capture particles (step 600d). Removal of the organelle-capture particles may occur by magnetic removal, centrifugation, filtration, or any combination thereof. Step 600d may result in a lysate depleted of one or more organelles. Contacting with a nucleic acid binding agent The workflow may include contacting the lysate with one or more nuclei-binding reagents (step 600e). The nuclei-binding reagent 640 may include a first epitope 642. The first epitope 642 may include, for example, a strong epitope described herein (e.g., biotin, fluorescein, and / or DIG). The nuclei-binding reagent 640 may be capable of specifically binding to one or more components of nuclei (e.g., nuclear envelope proteins), such as nuclear envelope surface component 610 (as shown in FIG. 6C). In some embodiments, the nuclei-binding reagent includes a carbohydrate-binding reagent (e.g., a carbohydrate-binding protein, a lectin). Contacting the nuclei with the nuclei-binding reagent may result in nuclei bound to the nuclei-binding reagent. The nuclei-binding reagent 640 may include a nuclei-indexing oligonucleotide 644. The nuclei-indexing oligonucleotide 644 may include a nuclei-indexing sequence (e.g., for sample indexing). The nuclei-indexing oligonucleotides 644 may remain associated with the nuclei throughout the workflow. In some embodiments, the workflow may include pooling nuclei from different samples once the nuclei are bound by a nuclei-binding reagent that includes a nuclei-indexing oligonucleotide.

[0232] Contacting with nuclei isolation particles and null particles The workflow may include contacting the lysate with nuclei isolation particles and null particles (step 600f). The nuclei isolation particles 650 may include a reagent capable of specifically binding to a nuclei-binding reagent described herein (e.g., first epitope-binding reagent 652 as shown in FIG. 6D). The first epitope-binding reagent 652 may be associated (e.g., immobilized, partially immobilized, encapsulated, partially encapsulated) with the nuclei isolation particles. The nuclei-binding reagent may include a primary antibody capable of specifically binding to one or more components of the nucleus, and the reagent capable of specifically binding to the nuclei-binding reagent may include a secondary antibody capable of specifically binding to the primary antibody. A plurality of barcodes 654 may be associated (e.g., immobilized, partially immobilized, encapsulated, partially encapsulated) with the nuclei isolation particles.

[0233] As shown in FIG. 6D , step 600f can result in nuclei bound to nuclei isolation particles via a reagent capable of specifically binding to the nuclei-binding reagent. In some embodiments, a population of nuclei within a sample can be separated from extranuclear cellular components according to the methods provided herein. In some embodiments, a population of nuclei within a sample can be individually separated (e.g., isolated) from one another according to the methods provided herein. Some embodiments of the compositions and methods disclosed herein may optionally be used to reduce or eliminate clumping of nuclei isolation particles. Some embodiments of the methods and compositions provided herein can be used, for example, to (1) reduce or eliminate binding of individual nuclei isolation particles to two or more nuclei and / or (2) reduce or eliminate binding of individual nuclei to multinuclei isolation particles.

[0234] In some embodiments, the percentage of nucleus isolation particles bound to a single nucleus or nucleus isolation particles not bound to a nucleus after the contacting step is 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% ,49%,50%,51%,52%,53%,54%,55%,56%,57%,58%,59%,60%,61%,62%,63%,64%,65%,66%,67%,68%,69%,70%,71%,72%,73%,74%,75%,76%,77%,78%,79%,80%,81%,82%,83%,84%,85%,86%,87%,88%,89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values, or about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 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%, 120%, 121%, 122%, 123%, The percentage may be 6%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or a number or range between any two of these values. In some embodiments, after the contacting step, the percentage of nucleus isolation particles bound to a single nucleus or nucleus isolation particles not bound to a nucleus is at least or at most 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 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% or 100%.

[0235] In some embodiments, the percentage of nuclei bound to a single nuclei isolation particle after the contacting step is 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values, or may be about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 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%, 120%, 121%, 122%, 123%, The percentage may be 6%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or a number or range between any two of these values. In some embodiments, the percentage of nuclei bound to a single nuclei isolation particle after the contacting step is at least or at most 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.0001%, 0.001%, 0.01%, 0.1%, 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% or 100%.

[0236] In some embodiments, the binding of a single nucleus to a nucleus isolation particle may follow a Poisson distribution. In some embodiments, the binding of a single nucleus to a nucleus isolation particle may follow a non-Poisson distribution. The probability that two distinct nuclei of a sample will bind to the same nucleus isolation particle is 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , or 10 -1 or higher, or at least 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , or 10 -1 The probability that two distinct nuclei from a sample can bind to the same nuclei isolation particle is at most 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , or 10 -1 The probability that a single nucleus can simultaneously bind to two or more distinct nucleus isolation particles is 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , or 10 -1 or higher, or at least 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , or 10 -1 The probability that a single nucleus can simultaneously bind to two or more separate nucleus isolation particles is up to 10 -6 , 10 -5 , 10 -4 , 10 -3 , 10 -2 , or 10 -1 Or it may be higher.

[0237] In some embodiments, the workflow includes contacting the lysate with nuclei isolation particles 650 and null particles 656. In some embodiments, the null particles 656 do not include multiple barcodes, magnetic properties, and / or reagents capable of specifically binding to nuclei-binding reagents (e.g., first epitope-binding reagents). The null particles may resemble nuclei isolation particles in all aspects except for the absence of multiple barcodes, magnetic properties, and / or reagents capable of specifically binding to nuclei-binding reagents (e.g., first epitope-binding reagents). In some embodiments, the lysate is contacted with null particles prior to contacting with the nuclei isolation particles. In some embodiments, the lysate is contacted with null particles and nuclei isolation particles simultaneously. Contacting with null particles can (1) reduce or eliminate binding of individual nuclei isolation particles to two or more nuclei and / or (2) reduce or eliminate binding of individual nuclei to multiple nuclei isolation particles.

[0238] In some embodiments, the ratio of nucleus isolated particles to null particles ranges from 1:100 to 100:1. In some embodiments, the ratio of nucleus isolated particles to null particles is up to 10:1. In some embodiments, the ratio of nucleus isolated particles to null particles is up to 100:1. In some embodiments, the ratio of nucleus isolated particles to null particles is up to 1:1000. In some embodiments, the ratio of nucleus isolated particles to null particles is at least 1:10. In some embodiments, the ratio of nucleus isolated particles to null particles is at least 1:100. In some embodiments, the ratio of nucleus isolated particles to null particles is at least 1:1000.

[0239] In some embodiments, the ratio of nuclei isolated particles to null particles is 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 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, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, :25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, It may be 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or a number or range between any two of the values, such as about 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:20, 1:21.5, 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, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35 , 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109, 1:110, 1:111, 1:112, 1:113, 1:114, 1:115, 1:116, 1:117 7, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1: 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, or a number or range between any two of the values. In some embodiments, the ratio of nuclei isolated particles to null particles is at least or at most 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3.5, 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, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34 4, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1: 65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109, 1:110, 1:111, 1:112, 1:113, 1:114, 1:115, 1:116, 1:117, 1:118, 1:119, 1:120, 1:121, 1:122, 1:123, 1:124, 1:125, 1:126, 1:127, 1:128, 1:129, 1:130, 1:131, 1:132, 1:133, 1:134, 1:135, 1:136, 1:137, 1:138, 1:139, 1:140, 1:141, 1:142, 1 The ratio may be 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000 or 1:10000.

[0240] Some embodiments of the methods and compositions provided herein use flow cytometry to (1) reduce or eliminate the binding of individual nuclei isolated particles to two or more nuclei and / or (2) reduce or eliminate the binding of individual nuclei to multiple nuclei isolated particles. For example, a series of droplets each containing a single nucleus and a series of droplets each containing a single nucleus can be combined to produce a series of droplets each containing a single nucleus and a single nuclei isolated particle. After the single nuclei bind to the single nuclei isolated particle within the droplets, each droplet in the series can be combined with another series of droplets containing the released first epitope described herein. After the first epitope binding site on the nuclei isolated particle is saturated, the multiple droplets can be pooled and subjected to downstream methods of the present disclosure.

[0241] Contacting with the liberated first epitope The workflow may include contacting the lysate with a liberated first epitope (step 600g). The liberated first epitope 660 may include all or a portion of the first epitope 642. The liberated first epitope may bind to an unbound first epitope-binding reagent associated with the nuclei isolation particles (e.g., first epitope-binding reagent 652 shown in FIG. 6E). After step 600g, most or all of the binding sites of the nuclei-binding reagent may be saturated (e.g., by binding of either the liberated first epitope or the first epitope of the nuclei-binding reagent), thereby reducing or preventing any additional binding of the nuclei isolation particles to nuclei. Contacting with the liberated first epitope may prevent aggregation of the nuclei isolation particles when separated from null particles downstream in the workflow.

[0242] In some embodiments, the ratio of released first epitope to nuclei isolated particles is 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 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, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:7 :24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 2:00, 2:01, 2:02, 2:03, 2:04, 2:05, 2:06, 2:07, 2:08, 2 4, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:910, 1:920, 1:930, 1:940, 1:950, 1:960, 1:970, 1:980, 1:990, 1:100, 1:1100, 1:1200, 1:1300, 1:1400, 1:1500, 1:1600, 1:1700, 1:1800, 1:1900, 1:2100, 1:2200, 1:2300, 1:2400, 1:2500, 1:2600, 1:2700, 1:2800, 1:2900, 1:3100, 1:3200, 1:3300, 1:3 :500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000 or a number or range between any two of the values, or about 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:20, 1:21.5, 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, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35 , 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1:65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109, 1:110, 1:111, 1:112, 1:113, 1:114, 1:115, 1:116, 1:117 7, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1: In some embodiments, the ratio of released first epitope to nuclei isolated particles may be at least 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:7000, 1:8000, 1:9000, 1:10000, or a number or range between any two of the values. In some embodiments, the ratio of released first epitope to nuclei isolated particles is at least or at most 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3.5, 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, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34 4, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43, 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55, 1:56, 1:57, 1:58, 1:59, 1:60, 1:61, 1:62, 1:63, 1:64, 1: 65, 1:66, 1:67, 1:68, 1:69, 1:70, 1:71, 1:72, 1:73, 1:74, 1:75, 1:76, 1:77, 1:78, 1:79, 1:80, 1:81, 1:82, 1:83, 1:84, 1:85, 1:86, 1:87, 1:88, 1:89, 1:90, 1:91, 1:92, 1:93, 1:94, 1:95, 1:96, 1:97, 1:98, 1:99, 1:100, 1:101, 1:102, 1:103, 1:104, 1:105, 1:106, 1:107, 1:108, 1:109, 1:110, 1:111, 1:112, 1:113, 1:114, 1:115, 1:116, 1:117, 1:118, 1:119, 1:120, 1:121, 1:122, 1:123, 1:124, 1:125, 1:126, 1:127, 1:128, 1:129, 1:130, 1:131, 1:132, 1:133, 1:134, 1:135, 1:136, 1:137, 1:138, 1:139, 1:140, 1:141, 1:142, 1 The ratio may be 1:96, 1:97, 1:98, 1:99, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000 or 1:10000.

[0243] In some embodiments, after the step of contacting with the free first epitope, the percentage of nuclei isolated particles comprising one or more unbound first epitope-binding reagents is 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 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%, 120%, 121%, 122%, 123%, 124%, 125%, 12 7%, 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%, It may be 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values, or about 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.001%, 0.01%, 0.1%, 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%, 120%, 121%, 122%, 123%, The percentage may be 6%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or a number or range between any two of these values. In some embodiments, after the step of contacting with the free first epitope, the percentage of nuclear isolated particles comprising one or more unbound first epitope binding reagents is at least or at most 0.000000001%, 0.00000001%, 0.0000001%, 0.000001%, 0.00001%, 0.0001%, 0.001%, 0.01%, 0.1%, 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%, It may be 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%.

[0244] Isolation of nuclear particles The workflow may include isolating nuclei isolation particles (step 600h). Isolating the nuclei isolation particles (and their associated nuclei) may include isolating the nuclei isolation particles by magnetic removal, filtration, centrifugation, or any combination thereof. Step 600h may produce a plurality of isolated nuclei isolation particles (each associated with one or zero nuclei) depleted of organelles and / or extranuclear cellular components.

[0245] Partitioning, nuclear lysis, and barcoding The workflow may include steps of dividing, nuclei lysis, and barcoding (step 600i). The multiple nuclei and their associated nuclei isolation particles may be divided into multiple compartments. In some embodiments, the multiple nuclei are separated from the nuclei isolation particles before dividing the nuclei into multiple compartments. The compartments may be microwells, droplets, or emulsions. One of the multiple compartments may contain a single nucleus derived from the multiple nuclei and its associated nuclei isolation particles. One of the multiple compartments may contain a single nucleus derived from the multiple nuclei and barcoded particles. One of the multiple compartments may contain a single nucleus derived from the multiple nuclei, its associated nuclei isolation particles, and barcoded particles. In some embodiments, the dimensions of the microwells (e.g., the depth of the microwells) are selected to optimize the trapping efficiency of the nuclei and nuclei isolation particles while also efficiently exchanging assay buffer and other reagents contained within the wells. Once divided, the nuclei may be lysed according to the methods provided herein. The barcoding step may include using a plurality of barcodes of the nucleus isolation particles and / or barcoded particles to generate a plurality of barcoded nucleus-indexing oligonucleotides and / or barcoded targets according to the methods provided herein. The product of step 600i (e.g., barcoded nucleus-indexing oligonucleotides and / or barcoded targets) may be subjected to downstream methods provided herein to generate a single-cell expression profile.

[0246] In some embodiments, one or more steps of the workflow include centrifugation. In some embodiments, one or more steps of the workflow do not include centrifugation. In some embodiments, none of the steps of the workflow include centrifugation. In some embodiments, the workflow does not include one or more of steps 600a, 600b, 600c, 600d, 600e, 600f, 600g, 600h, and / or 600i. For example, in some embodiments, the method does not include contacting with an organelle surface component binding reagent (step 600b), contacting with an organelle capture particle (step 600c), and / or removing the organelle capture particle (step 600d). Optionally, step 600a includes lysing the plurality of cells and lysing the organelle membrane without lysing the nuclear envelope, and therefore, steps 600b, 600c, and / or 600d are not performed. In some embodiments, organelle contaminants and / or extranuclear cellular component contaminants (e.g., mitochondrial contaminants) can be reduced (e.g., eliminated) for single-cell analysis (e.g., whole transcriptome analysis or proteomic analysis) without performing steps 600b, 600c, and / or 600d of the workflow. In some embodiments, one or more of steps 600a, 600b, 600c, 600d, 600e, 600f, 600g, 600h, and / or 600i are performed simultaneously. For example, the step of contacting with the organelle surface component-binding reagent (step 600b) and the step of contacting with the organelle-capture particle (step 600c) can occur simultaneously. In some such embodiments, the organelle surface component-binding reagent is associated with the organelle-capture particle. The organelle surface component binding reagent may be immobilized or partially immobilized on the organelle capture particle.For example, the organelle surface component binding reagent may be reversibly, irreversibly, covalently, non-covalently, or any combination thereof associated with the organelle capture particle. As another example, the organelle surface component binding reagent may be embedded, partially embedded, not embedded, encapsulated, partially encapsulated, not encapsulated, or a combination thereof in the organelle capture particle.

[0247] In some embodiments, the nucleus-binding reagent does not contain the first epitope. In some such embodiments, the nucleus-binding reagent may include a primary antibody capable of specifically binding to one or more components of the nucleus (e.g., a nuclear envelope protein), and the nucleus isolation particles are associated with a secondary antibody capable of specifically binding to the primary antibody. In some such embodiments, the free primary antibody (instead of the free first epitope) may be contacted with the lysate before isolating the nucleus isolation particles.

[0248] Example 2 Nuclei capture and barcoding workflow This example demonstrates a workflow for nuclei capture and barcoding. Figures 7A-7C are schematic diagrams of non-limiting exemplary workflows for nuclei isolation and single-cell profiling. The workflow may include cell lysis (e.g., using a photolysis buffer that dissolves the plasma membrane without lysing the nuclear membrane and / or one or more organelles). The workflow may include associating each nucleus with a nuclei-indexing oligonucleotide containing a nuclei-indexing sequence (e.g., for sample indexing). The workflow may include isolating nuclei from one or more cellular components and / or organelles. The workflow may include dividing individual nuclei into compartments and performing nuclei lysis. The workflow may include a step of barcoding (e.g., stochastically barcoding) target nucleic acid molecules and / or nuclear indexing oligonucleotides for single cell analysis (e.g., Rhapsody™ Assay (Becton, Dickinson and Company, Franklin Lakes, NJ), Chromium™ Single Cell 3' Solution (10X Genomics, San Francisco, CA)).

[0249] Sample Composition The sample 701 may include a plurality of cells 702. Samples for use in the disclosed methods may be those from which it is technically difficult or impossible to generate a single-cell suspension (frozen cells, fixed cells, tissue specimens, tumor specimens, epithelial tissue, formalin-fixed paraffin-embedded cells, and combinations thereof). The cells may include a plasma membrane 704 and a cell nucleus 706. The cell nucleus may include a nuclear envelope 708 and one or more nucleic acid target molecules 712. The nuclear envelope may include one or more nuclear envelope surface components 710, such as nuclear envelope proteins (e.g., lamins). The nucleus may include one or more target nucleic acid molecules (e.g., messenger RNA (mRNA), microRNA (miRNA), long non-coding RNA (long ncRNA or lncRNA), Piwi-interacting RNA (piRNA)). A cell may contain one or more organelles described herein (e.g., ribosomes, mitochondria), and one or more organelles may contain one or more organelle surface components. Mitochondria 716 may contain organelle surface components (e.g., mitochondrial surface components 718). A cell may also contain extranuclear cellular components 714. The extranuclear cellular components may include unwanted extranuclear cytoplasmic components (e.g., molecules that are unnecessary and / or deleterious to single-cell expression profiling). The extranuclear cellular components may include cytoplasmic fragments. The extranuclear cellular components may include organelles. The extranuclear cellular components may include mitochondria. The extranuclear cellular components may include ribosomes. The extranuclear cellular components may include rough endoplasmic reticulum. The extranuclear cellular components may include one or more unwanted nucleic acids (e.g., ribosomal RNA, mitochondrial DNA, mitochondrial RNA). The extranuclear cellular components may include one or more macromolecules that interfere with the single-cell expression analysis methods disclosed herein.The extranuclear cellular components 714 can include mitochondria, peroxisomes, cytosol, vesicles, lysosomes, plasma membranes, chloroplasts, intramitochondrial matrix, inner mitochondrial membrane, intermembrane space, outer mitochondrial membrane, secretory vesicles, smooth endoplasmic reticulum, rough endoplasmic reticulum, Golgi apparatus, phagosomes, endosomes, exosomes, plasma membranes, microtubules, microfilaments, intermediate filaments, filopodia, ruffles, lamellipodia, sarcomeres, focal adhesions, podosomes, ribosomes, microsomes, lipid rafts, cell wall, and combinations thereof.

[0250] Plasma membrane lysis The workflow may include plasma membrane lysis (step 700a) of a population of cells 702 of a sample 701. Plasma membrane lysis may produce a lysate containing multiple populations of nuclei, organelles, and extranuclear cellular components (e.g., ribosomes), as shown in FIG. 7A. The method may include lysing multiple cells without lysing the nuclear envelope. The method may include lysing multiple cells without lysing organelle membranes. The method may include lysing multiple cells and lysing organelle membranes without lysing the nuclear envelope. Plasma membrane lysis may be performed using a homogenizer (e.g., a Dounce homogenizer), detergent, or enzymatic methods. In some embodiments, the workflow includes contacting the lysate with an organelle surface component binding reagent, contacting the lysate with organelle capture particles, and / or removing the organelle capture particles (e.g., step 600b, step 600c, and / or step 600d of Example 1) before performing step 700b.

[0251] Contacting with a nucleic acid binding agent The workflow may include contacting the lysate with one or more nuclei-binding reagents (step 700b). The nuclei-binding reagent 740 may include a first epitope 742. The first epitope 742 may include a strong epitope (e.g., biotin, fluorescein, and / or DIG) described herein. The nuclei-binding reagent 740 may be capable of specifically binding to one or more components of nuclei (e.g., nuclear envelope proteins), such as nuclear envelope surface component 710 (as shown in FIG. 7B). In some embodiments, the nuclei-binding reagent includes a carbohydrate-binding reagent (e.g., a carbohydrate-binding protein, a lectin). Contacting the nuclei with the nuclei-binding reagent may result in nuclei bound to the nuclei-binding reagent. The nuclei-binding reagent 740 may include a nuclei-indexing oligonucleotide 744. The nuclei-indexing oligonucleotide 744 may include a nuclei-indexing sequence (e.g., for sample indexing). The nuclei-indexing oligonucleotides 744 may remain associated with the nuclei throughout the workflow. In some embodiments, the workflow may include pooling nuclei from different samples once the nuclei are bound by a nuclei-binding reagent that includes a nuclei-indexing oligonucleotide.

[0252] Contacting with nuclei isolated particles The workflow may include contacting the lysate with one or more nuclei isolation particles (step 700c). The nuclei isolation particles 750 may include a reagent capable of specifically binding to a nuclei-binding reagent described herein (e.g., first epitope-binding reagent 752 shown in FIG. 7B). The first epitope-binding reagent 752 may be associated with (e.g., immobilized, partially immobilized, encapsulated, partially encapsulated) the nuclei isolation particles through a cleavable linker 780. The cleavable linker 780 may operably link the first epitope-binding reagent 752 to the nuclei isolation particles 750. The cleavable linker may include a chemically cleavable linkage, a photocleavable linkage, an acid-labile linker, a heat-sensitive linkage, an enzymatically cleavable linkage, or a combination thereof. The nucleus-binding reagent may include a primary antibody capable of specifically binding to one or more components of the nucleus, and the reagent capable of specifically binding to the nucleus-binding reagent may include a secondary antibody capable of specifically binding to the primary antibody, wherein the secondary antibody is linked to the nucleus isolation particle through a cleavable linker. As shown in Figure 7B, step 700c may produce nuclei bound to the nucleus isolation particle by the reagent capable of specifically binding to the nucleus-binding reagent.

[0253] Isolation of nuclear particles The workflow may include isolating nuclei isolation particles (step 700d). Isolating the nuclei isolation particles (and associated nuclei) may include isolating the nuclei isolation particles by magnetic removal, filtration, centrifugation, or any combination thereof. Step 700d may produce a plurality of isolated nuclei isolation particles (and associated nuclei) dep...

Claims

1. 1. A method for determining the number of targets in a plurality of cells, comprising: isolating a plurality of nuclei of a plurality of cells using a nuclei isolation composition, wherein the nuclei isolation composition comprises a nuclei-binding reagent, the nuclei-binding reagent being capable of specifically binding to one or more components of a nucleus, and wherein isolating the plurality of nuclei comprises isolating nuclei bound to the nuclei-binding reagent using a reagent that can specifically bind to the nuclei-binding reagent, wherein the nuclei-binding reagent comprises a carbohydrate-binding reagent, wherein the carbohydrate-binding reagent comprises a carbohydrate-binding protein, wherein the carbohydrate-binding protein comprises a lectin; barcoding a plurality of targets in a plurality of nuclei using a plurality of barcodes to generate a plurality of barcoded targets, wherein each of the plurality of barcodes comprises a molecular beacon sequence and a target binding region, and wherein the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different sequences; obtaining sequencing data for a plurality of barcoded targets; estimating the number of each of the plurality of targets in the plurality of cells using molecular signature sequences of the plurality of barcodes in the sequencing data; A method comprising:

2. (a) isolating a plurality of nuclei comprises contacting a plurality of nuclei from a plurality of cells with a nuclei isolation composition to produce nuclei bound to a nuclei-binding agent; (b) the nucleic acid binding agent is associated with a first epitope; (c) the nucleic binding reagent is associated with a first epitope, and the reagent capable of specifically binding to the nucleic binding reagent comprises a first epitope-binding reagent; (d) the nucleic binding reagent is associated with a first epitope, and the reagent capable of specifically binding to the nucleic binding reagent comprises a first epitope-binding reagent, and the first epitope comprises biotin, a hapten, or a combination thereof; and / or (e) the nucleic binding reagent is associated with a first epitope, and the reagent capable of specifically binding to the nucleic binding reagent comprises a first epitope binding reagent, the first epitope comprising biotin, a hapten, or a combination thereof; and (i) the hapten comprises digoxigenin, 2,4-dinitrophenol, fluorescein, or a combination thereof; and / or (ii) the reagent capable of specifically binding to the nucleic acid-binding reagent comprises an anti-hapten antibody; or (iii) the reagent capable of specifically binding to the nucleic acid binding reagent comprises avidin, streptavidin, neutravidin, or a combination thereof; The method of claim 1.

3. 3. The method of claim 1 or 2, wherein the nucleobase binding reagent comprises a functional group selected from the group consisting of biotin, streptavidin, heparin, an aptamer, a click chemistry moiety, digoxigenin, a primary amine, a carboxyl, a hydroxyl, an aldehyde, a ketone, and combinations thereof.

4. 4. The method of claim 1, wherein the reagent capable of specifically binding to the nucleic-binding reagent comprises a functional group selected from the group consisting of biotin, streptavidin, heparin, an aptamer, a click chemistry moiety, digoxigenin, a primary amine, a carboxyl, a hydroxyl, an aldehyde, a ketone, and combinations thereof.

5. (a) the lectin comprises a mannose-binding lectin, a galactose-binding lectin, an N-acetylgalactosamine-binding lectin, an N-acetylglucosamine-binding lectin, an N-acetylneuraminic acid-binding lectin, a fucose-binding lectin, or a combination thereof; or (b) the lectin comprises concanavalin A (ConA), lentil lectin (LCH), snowdrop lectin (GNA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA), jacalin (AIL), hairy vetch lectin (VVL), wheat germ agglutinin (WGA), elderberry lectin (SNA), Maackia amurensis leukoagglutinin (MAL), Maackia amurensis hemagglutinin (MAH), Ulex europaeus agglutinin (UEA), Aleuria aurantia lectin (AAL), or a combination thereof; The method according to any one of claims 1 to 4.

6. a nucleus binding agent is associated with the nucleus isolation particle; and (a) a reagent capable of specifically binding to a nuclei-binding reagent is associated with the nuclei-isolated particle; (b) a reagent capable of specifically binding to the nucleus-binding reagent is associated with the nucleus isolation particle, and a reagent capable of specifically binding to the nucleus-binding reagent is immobilized on the nucleus isolation particle; (c) a reagent capable of specifically binding to the nucleus-binding reagent is associated with the nucleus-isolated particle through a cleavable linker; (d) a reagent capable of specifically binding to the nucleus-binding reagent is associated with the nucleus-isolated particle through a cleavable linker, the cleavable linker comprising a chemically cleavable linkage, a photocleavable linkage, an acid-labile linker, a heat-sensitive linkage, an enzymatically cleavable linkage, or a combination thereof; (e) the nucleus isolation particles comprise nucleus isolation beads; (f) the nucleus isolation particles 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, hydrogel beads, or any combination thereof; (g) the nucleus isolation particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; (h) the nucleus isolated particle is disintegrable; (i) the nucleus-isolated particles comprise nucleus-isolated disintegrable hydrogel particles; and / or (j) isolating the nuclei bound to the nuclei-binding reagent comprises contacting the nuclei bound to the nuclei-binding reagent with a plurality of nuclei isolation particles; The method according to any one of claims 2 to 5.

7. (a) isolating the nuclei bound to the nuclei-binding reagent comprises isolating the nuclei-isolated particles by magnetic removal, centrifugation, or any combination thereof; (b) isolating the nuclei bound to the nuclei-binding reagent comprises isolating the nuclei-isolated particles by magnetic removal, centrifugation, or any combination thereof; and prior to the step of isolating the plurality of nuclei isolated particles, contacting the plurality of nuclei bound to the nuclei isolated particles with a liberated first epitope, wherein the liberated first epitope comprises all or a portion of the first epitope; (c) a plurality of barcodes are associated with the nuclear isolation particle; (d) at least one barcode of the plurality of barcodes is immobilized on a nuclei isolation particle; (e) at least one barcode of the plurality of barcodes is partially immobilized on the nuclei isolation particle; (f) at least one barcode of the plurality of barcodes is encapsulated in a nuclei isolation particle; and / or (g) at least one barcode of the plurality of barcodes is partially encapsulated in a nucleus isolation particle; The method of claim 6.

8. prior to isolating a plurality of nuclei of a plurality of cells using the nucleus isolation composition, lysing the plasma membrane of a plurality of cells; depleting one or more organelles of the plurality of cells using an organelle-capturing composition comprising an organelle-binding reagent, the organelle-binding reagent capable of specifically binding to one or more components of one or more organelles of the plurality of cells; The method according to any one of claims 1 to 7, comprising:

9. One or more of the following may apply: (a) depleting one or more organelles comprises contacting one or more organelles of a plurality of cells with an organelle-capturing composition to produce one or more organelles bound to an organelle component-binding reagent; (b) depleting one or more organelles comprises contacting one or more organelles of the plurality of cells with an organelle-capturing composition to produce one or more organelles bound to an organelle component-binding reagent; depleting the one or more organelles comprises depleting the one or more organelles bound to the organelle-binding reagent using a reagent capable of specifically binding to the organelle-binding reagent; (c) depleting one or more organelles comprises contacting one or more organelles of the plurality of cells with an organelle-capturing composition to produce one or more organelles bound to an organelle component-binding reagent; depleting the one or more organelles comprises depleting the one or more organelles bound to the organelle-binding reagent using a reagent capable of specifically binding to the organelle-binding reagent; the reagent capable of specifically binding to the organelle-binding reagent comprises a functional group selected from the group consisting of biotin, streptavidin, heparin, an aptamer, a click chemistry moiety, digoxigenin, a primary amine, a carboxyl, a hydroxyl, an aldehyde, a ketone, and any combination thereof; (d) the organelle-binding reagent is associated with a second epitope, and the reagent capable of specifically binding to the organelle-binding reagent comprises a second epitope-binding reagent; (e) the organelle-binding reagent is associated with a second epitope, and the reagent capable of specifically binding to the organelle-binding reagent comprises a second epitope-binding reagent, wherein the second epitope comprises biotin, a hapten, or a combination thereof; (f) the organelle-binding reagent is associated with a second epitope, and the reagent capable of specifically binding to the organelle-binding reagent comprises a second epitope-binding reagent, the second epitope comprising biotin, a hapten, or a combination thereof, and the hapten comprising digoxigenin, 2,4-dinitrophenol, fluorescein, or a combination thereof; (g) the reagent capable of specifically binding to the organelle-binding reagent comprises an anti-hapten antibody; (h) the reagent capable of specifically binding to the organelle-binding reagent comprises an anti-hapten antibody; The reagent capable of specifically binding to the organelle-binding reagent comprises avidin, streptavidin, neutravidin, or a combination thereof; (i) the organelle-binding reagent comprises a functional group selected from the group consisting of biotin, streptavidin, heparin, an aptamer, a click chemistry moiety, digoxigenin, a primary amine, a carboxyl, a hydroxyl, an aldehyde, a ketone, and any combination thereof; and (j) a reagent capable of specifically binding to the organelle-binding reagent is associated with the organelle-capture particle; The method of claim 8.

10. A method comprising (a) or (d) of claim 9, the organelle-binding reagent comprises a primary antibody capable of specifically binding to one or more components of one or more organelles of the plurality of cells; 10. The method of claim 9, wherein the reagent capable of specifically binding to the organelle-binding reagent comprises a secondary antibody capable of specifically binding to the primary antibody.

11. A method comprising (e) of claim 9, (a) a reagent capable of specifically binding to an organelle-binding reagent is immobilized on an organelle-capturing particle; (b) the organelle-capturing particles include organelle-capturing beads; (c) the organelle capture particles 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; (d) the organelle-capturing particle comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; and / or (e) depleting organelles from the plurality of cells using the organelle-capture composition includes depleting one or more organelle-capture particles by magnetic removal, centrifugation, or any combination thereof; 10. The method of claim 9.

12. (a) the organelle contains mitochondria from multiple cells; and / or (b) one or more components of one or more organelles of the plurality of cells comprises ABCD3, ESR2, NOS3, ALB, HIF1A, NR3C1, ATP5A1, HK1, PGR, CASQ1, HSPA1A, PHB, CLTC, HSPD1, PLN, COX4I1, IFM1, SOD1, CPS1, LGALS3, TP53, cytochrome C oxidase, MAPT, TP5B, ERN1, MT-CO1, VDAC1, or a combination thereof; and / or (c) the organelle-binding reagent comprises an organelle surface component-binding reagent, wherein one or more components of one or more organelles comprise one or more organelle surface components, and the organelle-binding reagent is capable of specifically binding to the one or more organelle surface components; The method according to any one of claims 8 to 11.

13. Multiple cells (i) a tissue sample; (ii) an epithelial tissue sample; (iii) frozen cells; (iv) fixed cells, (v) formalin-fixed paraffin-embedded cells; (vi) tumor cells, (vii) fixed tumor cells; (viii) frozen tumor cells, and / or (ix) formalin-fixed paraffin-embedded tumor cells; The method according to any one of claims 1 to 12, comprising:

14. a nucleus isolation composition comprising a nucleus binding reagent, wherein the nucleus binding reagent is capable of specifically binding to one or more components of a nucleus, the nucleus binding reagent comprises a carbohydrate binding reagent, the carbohydrate binding reagent comprises a carbohydrate binding protein, the carbohydrate binding protein comprises a lectin; and a plurality of barcodes, each of the plurality of barcodes comprising a molecular beacon sequence and a target binding region, wherein the molecular beacon sequences of at least two barcodes of the plurality of barcodes comprise different sequences.

15. One or more of the following may apply: (a) a nucleus-binding agent is associated with the nucleus-isolated particle; (b) a nucleus-binding reagent is associated with the nucleus-isolation particle, and a reagent capable of specifically binding to the nucleus-binding reagent is associated with the nucleus-isolation particle; (c) a nucleus-binding reagent is associated with the nucleus isolation particle, and a reagent capable of specifically binding to the nucleus-binding reagent is associated with the nucleus isolation particle, and a reagent capable of specifically binding to the nucleus-binding reagent is immobilized on the nucleus isolation particle; (d) a nucleus-binding reagent is associated with the nucleus-isolated particle; a reagent capable of specifically binding to the nucleus-binding reagent is associated with the nucleus-isolated particle; the reagent capable of specifically binding to the nucleus-binding reagent is associated with the nucleus-isolated particle through a cleavable linker; the cleavable linker comprises a chemically cleavable linkage, a photocleavable linkage, an acid-labile linker, a heat-sensitive linkage, an enzymatically cleavable linkage, or a combination thereof; (e) the nucleus isolation particles comprise nucleus isolation beads; (f) the nucleus isolation particles 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, hydrogel beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; (g) the nucleus isolation particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; (h) the nucleus isolated particle is disintegrable; (i) the nucleus-isolated particles comprise nucleus-isolated disintegrable hydrogel particles; (j) a plurality of barcodes are associated with the nuclear isolation particle; (k) at least one barcode of the plurality of barcodes is immobilized on a nuclei isolation particle; (l) at least one barcode of the plurality of barcodes is partially immobilized on a nuclei isolation particle; (m) at least one barcode of the plurality of barcodes is encapsulated in a nucleus isolation particle; (n) at least one barcode of the plurality of barcodes is partially encapsulated in a nucleus isolation particle; (o) the one or more components of the nucleus include lamin, emerin, nesprin, nurim, UNC-83, kral, ZYG-12, Kms1p, UNC-84, kraloid, SUN-1, Sad1p, LBR, MAN1, LAP1, LAP2, LINK, nuclear pore complex, a portion thereof, or a combination thereof; (p) one or more components of the core include a sugar, an oligosaccharide, a polysaccharide, a derivative thereof, or a combination thereof; (q) one or more components of the core comprise a monosaccharide, a disaccharide, a polyol, a malto-oligosaccharide, a non-malto-oligosaccharide, a starch, a non-starch polysaccharide, a derivative thereof, or a combination thereof; (r) one or more components of the core include glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, maltodextrin, raffinose, stachyose, fructooligosaccharides, amylose, amylopectin, modified starch, glycogen, cellulose, hemicellulose, pectin, hydrocolloids, derivatives thereof, or combinations thereof; (s) one or more components of the core are selected from the group consisting of α-D-mannosyl residues, α-D-glucosyl residues, branched α-mannosidic structures of high α-mannose types, branched α-mannosidic structures of hybrid and biantennary complex N-glycans, fucosylated core regions of biantennary and triantennary complex N-glycans, α1-3 and α1-6 linked high mannose structures, Galβ1-4GalNAcβ1-R, Galβ1-3GalNAcα1-Ser / Thr, (Sia)Galβ1-3GalNAcα1-Ser / Thr, GalNAcα-Ser / Thr, GlcNAcβ1-4GlcNAcβ1-4GlcNAc, Neu5Ac (sialic acid), Neu5Acα2-6Gal(NAc)-R, Neu5A c / Gcα2,3Galβ1,4Glc(NAc), Neu5Ac / Gcα2,3Galβ1,3(Neu5Acα2,6)GalNAc, Fucα1-2Gal-R, Fucα1-2Galβ1-4(Fucα1-3 / 4)Galβ1-4GlcNAc, R2-GlcNAcβ1-4(Fucα1-6)GlcNAc-R1, derivatives thereof, or combinations thereof, (t) one or more components of the core include a glycoprotein, a glycolipid, or a combination thereof; (u) the nuclear binding reagent comprises a nuclear envelope surface component binding reagent, wherein the nuclear binding reagent is capable of specifically binding to one or more nuclear envelope surface components; (v) the composition comprises an organelle-capturing composition comprising an organelle-binding reagent, the organelle-binding reagent capable of specifically binding to one or more components of one or more organelles; and (w) the composition comprises an organelle capture composition comprising an organelle-binding reagent and a reagent capable of specifically binding to the organelle-binding reagent, wherein the organelle-binding reagent is capable of specifically binding to one or more components of one or more organelles; The bar-coded composition of claim 14.

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