Massively parallel single cell analysis

Abstract

The disclosure provides for methods, compositions, and kits for multiplex nucleic acid analysis of single cells. The methods, compositions and systems may be used for massively parallel single cell sequencing. The methods, compositions and systems may be used to analyze thousands of cells concurrently. The thousands of cells may comprise a mixed population of cells (e.g., cells of different types or subtypes, different sizes).

1 - 26 . (canceled) 
     
     
         27 . A compartment, comprising:
 a single particle associated with a plurality of oligonucleotides, wherein each of the plurality of oligonucleotides comprises a cellular label sequence, a molecular label sequence, and a target-binding region, wherein the cellular label sequence of each of the plurality of oligonucleotides is the same, and wherein at least 100 of the plurality of oligonucleotides comprise different molecular label sequences.   
     
     
         28 . The compartment of  claim 27 , wherein the target-binding region comprises an oligo-dT sequence, a gene-specific sequence, a random multimer sequence, or a combination thereof. 
     
     
         29 . The compartment of  claim 27 , wherein each of the plurality of oligonucleotides further comprises at least one of (a) a sample label sequence and (b) a universal label sequence. 
     
     
         30 . The compartment of  claim 27 , wherein the molecular label sequences comprise random sequences. 
     
     
         31 . The compartment of  claim 27 , wherein at least 10,000 of the plurality of oligonucleotides comprise different molecular label sequences. 
     
     
         32 . The compartment of  claim 27 , wherein the plurality of oligonucleotides comprises at least 100,000 oligonucleotides. 
     
     
         33 . The compartment of  claim 27 , wherein the compartment:
 comprises a single cell or a lysate of a single cell; and/or   is a microwell or a droplet.   
     
     
         34 . The compartment of  claim 33 , wherein the single cell is a rare cell, a cancer cell, an immune cell, a cell from a tissue, a human cell, a cell comprising viral polynucleotides, or a combination thereof. 
     
     
         35 . The compartment of  claim 33 , wherein the plurality of oligonucleotides is capable of labeling individual occurrences of target molecules of the single cell or the lysate of the single cell. 
     
     
         36 . The compartment of  claim 35 , wherein said target molecules comprise a DNA molecule, an RNA molecule, or a combination thereof. 
     
     
         37 . The compartment of  claim 33 , wherein the plurality of oligonucleotides is capable of labeling individual occurrences of mRNA molecules of the single cell or the lysate of the single cell via reverse transcription. 
     
     
         38 . The compartment of  claim 27 , wherein the compartment is a compartment of a microtiter plate or a microfluidic chip. 
     
     
         39 . The compartment of  claim 33 , wherein the diameter of the microwell:
 is from 1-fold to 2-fold the diameter of said single particle;   is about equal to the sum of the diameter of said single particle and the diameter of said single cell; and/or   is at least 10 micrometers.   
     
     
         40 . The compartment of  claim 33 , wherein the depth of the microwell:
 is about equal to the sum of the diameter of said single particle and the diameter of said single cell; and/or   is at least 1-fold the diameter of said single particle.   
     
     
         41 . The compartment of  claim 33 , wherein said droplet is in an emulsion. 
     
     
         42 . The compartment of  claim 33 , wherein the volume of said droplet is between one picoliter and one microliter. 
     
     
         43 . The compartment of  claim 33 , wherein said droplet is an aqueous droplet within a continuous oil phase, or an oil droplet within a continuous aqueous phase, or a droplet within a larger droplet. 
     
     
         44 . The compartment of  claim 27 , wherein the particle comprises polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, silica gel, controlled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose, cellulose, or a combination thereof. 
     
     
         45 . The compartment of  claim 27 , wherein the particle is a microparticle or a nanoparticle. 
     
     
         46 . The compartment of  claim 27 , wherein the particle is a bead, and wherein the bead is a streptavidin bead, an agarose bead, a magnetic beads, a hydrogel bead, an antibody conjugated bead, a protein A conjugated bead, a protein G conjugated bead, a protein NG conjugated bead, a protein L conjugated bead, an oligodT conjugated bead, a silica bead, a silica-like bead, an anti-biotin microbead, or an anti-fluorochrome microbead.