Methods of identifying multiple epitopes in cells
The invention provides methods, compositions, kits and devices for the detection of target molecules. In some embodiments, the invention allows for multiplexed target molecule detection.
1. A method comprising:
(a) binding an agent to a target molecule in a population of cells in a first reaction volume, wherein the agent comprises a target-specific nucleic acid barcode that identifies a corresponding target molecule;
(b) separating the cells into a plurality of second reaction volumes, wherein at least some of the second reaction volumes receive a single cell from the population of cells; and
(c) adding a cell-specific nucleic acid barcode or complement thereof to the target-specific nucleic acid barcode or complement thereof in the second reaction volumes.
2. The method of claim 1 , wherein the method further comprises:
(d) pooling a plurality of the second reaction volumes after step (c).
3. The method of claim 2 , wherein the method further comprises:
(e) sequencing the product of step (c) after pooling.
4. The method of claim 1 , wherein the cells of (a) are fixed and/or permeabilized.
5. The method of claim 1 , wherein the cells are lysed between (b) and (c).
6. The method of claim 1 , wherein the adding of (c) is done via hybridization, ligation, primer extension, or primer extension followed by ligation.
7. The method of claim 1 , wherein the second reaction volumes of (b) are droplets, microdroplets, wells, microwells, microbubbles, or enclosures in a microfluidics device.
8. The method of claim 1 , wherein (c) is done by:
combining at least some of the second reaction volumes with a cell-specific nucleic acid barcode, wherein the second reaction volumes receive different cell-specific nucleic acid barcodes; and
incubating the second reaction volumes under conditions sufficient to link the cell-specific nucleic acid barcode or complement thereof to the target-specific nucleic acid barcode or complement thereof.
9. The method of claim 8 , wherein a nucleic acid comprising the cell-specific nucleic acid barcode or a nucleic acid comprising the target-specific nucleic acid barcode further comprises a random sequence.
10. The method of claim 1 , wherein the target molecule is a protein.
11. The method of claim 1 , wherein the target molecule is a nucleic acid.
12. The method of claim 11 , wherein the target molecule is RNA or complementary DNA (cDNA).
13. The method of claim 1 , wherein the target molecule is intracellular.
14. The method of claim 1 , wherein the target molecule is on the surface of the cells.
15. The method of claim 1 , wherein the agent is an antibody-oligonucleotide conjugate.
16. The method of claim 1 , wherein the agent is an oligonucleotide, an oligonucleotide probe, a primer or an aptamer.
17. The method of claim 1 , wherein:
step (a) comprises binding a plurality of agents to a plurality of target molecules in the population of cells, wherein each agent comprises a target-specific nucleic acid barcode that identifies the target molecule to which the agent binds, and
step (c) comprises adding a cell-specific nucleic acid barcode or complement thereof to the target-specific nucleic acid barcodes or complements thereof in the second reaction volumes.
18. The method of claim 1 , wherein the cell-specific nucleic acid barcode or complement thereof and the target-specific nucleic acid barcode or complement thereof are added together on a bead.
19. The method of claim 1 , wherein the cell-specific nucleic acid barcode comprises a series of monomeric subunits that forms a code that is associated with a specific cell.
20. The method of claim 1 , wherein the population of cells of (a) is contained in a well of a multi-well plate.
21. The method of claim 20 , wherein the method is performed on a plurality of populations of cells each contained in a well of a microtiter plate.