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 of associating assayable oligonucleotide subunits with nucleic acids that are in or on cells or cell compartments, comprising:
linking assayable oligonucleotide subunits to nucleic acids that are in or on the cells or cell compartments by a split-and-pool process that comprises a first round of assayable oligonucleotide subunit addition and a second round of assayable oligonucleotide subunit addition, wherein the assayable oligonucleotide subunits are added sequentially to the nucleic acids in the first and second rounds.
2. The method of claim 1 , wherein three or more assayable oligonucleotide subunits are sequentially added to the nucleic acids.
3. The method of claim 1 , wherein the assayable oligonucleotide subunits added to the nucleic acids become part of a cell origination barcode.
4. The method of claim 1 , wherein the method comprises adding a random sequence to the nucleic acids in the first round of addition, the second round of addition, or a subsequent round of assayable oligonucleotide subunit addition.
5. The method of claim 1 , further comprising directly or indirectly adding a biotin to the nucleic acids.
6. The method of claim 5 , further comprises enriching for biotinylated nucleic acids.
7. The method of claim 1 , further comprising sequencing the nucleic acids, or an amplification product thereof, after the assayable oligonucleotide subunits have been added.
8. The method of claim 1 , wherein the nucleic acids comprise cellular nucleic acids.
9. The method of claim 1 , wherein the nucleic acids comprise genomic DNA.
10. The method of claim 1 , wherein the nucleic acids comprise RNA.
11. The method of claim 1 , wherein the nucleic acids comprise cDNA.
12. The method of claim 1 , wherein the nucleic acids comprise methylated nucleic acids.
13. The method of claim 12 , further comprising determining the methylation state of the nucleic acids.
14. The method of claim 1 , wherein the nucleic acids are oligonucleotides.
15. The method of claim 14 , wherein the nucleic acids are oligonucleotide probes that are directly or indirectly hybridized to nucleic acids in the cells or cell compartments.
16. The method of claim 14 , wherein the oligonucleotides are bound to proteins.
17. The method of claim 14 , wherein the oligonucleotides are conjugated to antibodies.
18. The method of claim 1 , wherein the nucleic acids are in the cells or cell compartments.
19. The method of claim 1 , wherein the cells or cell compartments are fixed and/or permeabilized.
20. The method of claim 1 , wherein the nucleic acids are on the cells or cell compartments.
21. The method of claim 1 , wherein the linking is done by ligation, gap-fill/ligation, by extension by a polymerase, hybridization or affinity binding.
22. The method of claim 21 , wherein the ligation is splinted using a bridging oligonucleotide.
23. The method of claim 1 , wherein the linking is done by extension by a polymerase.
24. The method of claim 1 , wherein the method is performed using a cell suspension.
25. The method of claim 1 , wherein method is performed using cultured cells, blood cells, cells from a tissue, or cell compartments isolated therefrom.
26. The method of claim 1 , wherein the method is performed using cells or cell compartments that have been exposed to an agent or genetically modified.
27. The method of claim 1 , wherein the method is performed using cell compartments.
28. The method of claim 1 , wherein the cell or cell compartments subjected to the first round of assayable oligonucleotide subunit addition correspond to a single biomolecular sample or each sample of a plurality of multiple biomolecular samples.