Compositions and methods for cellular processing
Provided herein are compositions and methods for cellular analysis. Nucleic acid from single cells may be processed in one or more partitions. A partition may comprise a cell bead and one or more enzymes for nucleic acid processing. A partition may comprise a functionalized polymer. In some cases, single cells may be subjected to epigenetic analysis, thereby generating an epigenetic profile for each cell from a plurality of cells.
1. A method comprising:
(a) providing a cell bead comprising (i) a nucleus and (ii) a nuclease coupled to a ligand;
(b) using said nuclease to cleave chromatin in said nucleus, thereby generating a cleaved nucleic acid molecule;
(c) partitioning (i) said cell bead, (ii) a plurality of nucleic acid barcode molecules, and (iii) a functionalized polymer to generate a partition, wherein said functionalized polymer is selected from the group consisting of dextran, polyethylene glycol (PEG), and polyacrylamide;
(d) outside of said cell bead in said partition, using said functionalized polymer to bind to said nuclease via said ligand; and
(e) using said cleaved nucleic acid molecule or a derivative thereof and a nucleic acid barcode molecule of said plurality of nucleic acid barcode molecules to perform one or more reactions in said partition to generate a barcoded nucleic acid molecule.
2. The method of claim 1 , wherein said partition is a droplet or a well.
3. The method of claim 1 , wherein said one or more reactions are performed outside said cell bead.
4. The method of claim 1 , wherein said one or more reactions comprise nucleic acid extension, nucleic acid amplification, or nucleic acid ligation.
5. The method of claim 1 , wherein said cell bead is functionalized.
6. The method of claim 1 , wherein said functionalized polymer is positively charged or negatively charged.
7. The method of claim 1 , wherein said partition comprises a cation for activating said nuclease.
8. The method of claim 7 , wherein said cation is magnesium or calcium.
9. The method of claim 1 , wherein said nuclease is micrococcal nuclease (MNase) or a deoxyribonuclease (DNase).
10. The method of 1 , wherein said nuclease is an engineered nuclease.
11. The method of claim 10 , further comprising binding said ligand of said engineered nuclease to a binding partner coupled to said functionalized polymer, wherein said binding partner inhibits activity of said engineered nuclease.
12. The method of claim 1 , wherein said nucleic acid barcode molecule is attached to a bead.
13. The method of claim 1 , wherein said nucleic acid barcode molecule is attached to a bead, wherein said bead is a gel bead.
14. The method of claim 1 , wherein said functionalized polymer comprises a linear polymer or a branched polymer.
15. The method of claim 1 , wherein said functionalized polymer is not cross-linked.
16. The method of claim 1 , wherein said partition further comprises a magnetic particle.
17. The method of claim 16 , wherein said magnetic particle comprises, attached thereto, a nucleic acid molecule comprising a capture sequence.
18. The method of claim 17 , wherein said capture sequence is a poly-T sequence, a random N-mer sequence, or a targeted capture sequence.
19. The method of claim 1 , wherein said functionalized polymer is incapable of diffusing into said cell bead.