Analysis of nucleic acids associated with single cells using nucleic acid barcodes
Provided herein are methods and compositions for analyzing nucleic acids associated with single cells using nucleic acid barcodes. According to some embodiments, a method for producing one or more polynucleotides of interest comprises: obtaining a plurality of RNAs associated with one or more samples, wherein the samples are obtained from one or more subjects, each RNA is associated with a single sample, and the RNAs associated with each sample are present in a separate reaction volume; adding an adapter molecule to the RNAs associated with each sample, wherein the adapter molecule is generated using an enzymatic reaction and comprises a universal priming sequence, a barcode sequence, and a binding site; and incorporating the barcode sequence into one or more polynucleotides associated with each sample, thereby producing the one or more polynucleotides of interest.
1. A method of attaching a polynucleotide to a solid support, wherein the polynucleotide contains an RNA polymerase (RNAP) promoter and a barcode sequence, the method comprising the steps of:
a) generating a hydrophilic compartment of an inverse emulsion, the hydrophilic compartment containing:
a solid support,
a barcode oligonucleotide comprising the RNAP promoter and the barcode sequence, and
an oligonucleotide bound to a surface of the solid support via a capture moiety; and
b) performing a polymerase extension reaction to incorporate the RNAP promoter and barcode sequence into the bound oligonucleotide on the solid support.
2. The method of claim 1 , wherein the barcode oligonucleotide further comprises a 5′ sequence identical or complementary to a PCR reverse primer sequence.
3. The method of claim 2 , further comprising performing a PCR reaction to amplify the barcode oligonucleotide.
4. The method of claim 1 , wherein the solid support is a bead or a surface.
5. The method of claim 1 , wherein the capture moiety is streptavidin.
6. The method of claim 1 , wherein the capture moiety comprises a carboxyl group, epoxy group, or hydroxyl group.
7. The method of claim 1 , wherein the capture moiety comprises gold to capture thiolyated oligonucleotides.
8. The method of claim 1 , wherein the barcode oligonucleotide further comprises a universal priming sequence and a binding site.
9. The method of claim 8 , wherein the barcode oligonucleotide comprises an RNAP promoter selected from the group consisting of T7, T3, and SP6.
10. The method of claim 8 , wherein the barcode oligonucleotide further comprises a nicking endonuclease restriction site selected from the group consisting of Nt.BbvCI, Nt.BspQI, Nt.BsmAI, Nt.BstNBI, Nt.AlwI, and Nt.BsmAI.
11. The method of claim 8 , wherein the binding site is one or more G nucleotides.
12. A solid support attached to a polynucleotide, wherein the polynucleotide contains an RNAP promoter and a barcode sequence.
13. The solid support of claim 12 , wherein the solid support is a bead or a surface.
14. The solid support of claim 12 , wherein the polynucleotide is attached to a surface of the solid support via a capture moiety.
15. The solid support of claim 14 , wherein the capture moiety is streptavidin.
16. The solid support of claim 14 , wherein the capture moiety comprises a carboxyl group, epoxy group, or hydroxyl group.
17. The solid support of claim 14 , wherein the capture moiety comprises gold to capture thiolyated oligonucleotides.
18. The solid support of claim 12 , wherein the polynucleotide further comprises a universal priming sequence and a binding site.
19. The solid support of claim 12 , wherein the polynucleotide comprises an RNAP promoter selected from the group consisting of T7, T3, and SP6.
20. The solid support of claim 18 , wherein the polynucleotide further comprises a nicking endonuclease restriction site selected from the group consisting of Nt.BbvCI, Nt.BspQI, Nt.BsmAI, Nt.BstNBI, Nt.AlwI, and Nt.BsmAI.
21. The solid support of claim 18 , wherein the binding site is one or more G nucleotides.
22. The method of claim 1 , wherein the bound oligonucleotide comprises a 3′ sequence complementary to a 3′ sequence of the barcode oligonucleotide.
23. The method of claim 3 , further comprising using a fluorophore-labeled reverse primer.
24. The method of claim 1 , wherein at least 10, 30, 100, 300, 1,000, 3,000, 10,000, 30,000, 100,000, 300,000, or 1,000,000 copies of the barcode sequence are bound to the solid support.
25. The method of claim 1 , wherein at most or on average one barcode oligonucleotide is present per hydrophilic compartment.