Compositions and methods for cellular processing

Patent metadata

Field	Value
Publication number	US-12065688-B2
Application number	US-201916434095-A
Country	US
Kind code	B2
Filing date	Jun 6, 2019
Priority date	Aug 20, 2018
Publication date	Aug 20, 2024
Grant date	Aug 20, 2024

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• 10X Genomics Inc

Inventors

• Bell Jason

Classifications

• C12Y301/31001

  Micrococcal nuclease (3.1.31.1) · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title