GENE EXPRESSION ANALYSIS IN SINGLE CELLS

• US 20120010091A1
• Filed: 03/23/2010
• Published: 01/12/2012
• Est. Priority Date: 03/30/2009
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Linnarson, Sten
• Application Number:
  US13/255,433
• Publication Number:
  US 20120010091A1
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

The present invention provides methods and compositions for the analysis of gene expression in single cells or in a plurality of single cells. The invention provides methods for preparing a cDNA library from individual cells by releasing mRNA from each single cell to provide a plurality of individual mRNA samples, synthesizing cDNA from the individual mRNA samples, tagging the individual cDNA, pooling the tagged cDNA samples and amplifying the pooled cDNA samples to generate a cDNA library. The invention also provides a cDNA library produced by the methods described herein. The invention farther provides methods for analyzing gene expression in a plurality of cells by preparing a cDNA library as described herein and sequencing the library.

Claims

1. A method of preparing a cDNA library from a plurality of single cells, the method comprising the steps of:

releasing mRNA from each single cell to provide a plurality of individual mRNA samples, wherein the mRNA in each individual mRNA sample is from a single cell;

synthesizing a first strand of cDNA from the mRNA in each individual mRNA sample and incorporating a tag into the cDNA to provide a plurality of tagged cDNA samples, wherein the cDNA in each tagged cDNA sample is complementary to mRNA from a single cell;

pooling the tagged cDNA samples; and

amplifying the pooled cDNA samples to generate a cDNA library comprising double-stranded cDNA.

2. The method according to claim 1, wherein in step (ii) the tag is incorporated into the cDNA during its synthesis.

3. The method according to claim 1, wherein synthesis of the first strand of cDNA in step (ii) is directed by a cDNA synthesis primer (CDS) that includes an RNA complementary sequence (RCS) that is at least partially complementary to one or more mRNA in an individual mRNA sample.

4. The method according to claim 3, wherein the RCS is at least partially complementary to a portion of the first strand of cDNA, such that it is able to direct the synthesis of a second strand of cDNA using the first strand of cDNA as a template.

5. The method according to claim 3, wherein a template-switching oligonucleotide (TSO) is added to each individual mRNA sample, wherein said TSO comprises a portion which is at least partially complementary to a portion at the 3′

end of the first strand of cDNA.

6. The method according to claim 1, wherein the first strand of cDNA includes a 3′

portion comprising a plurality of cytosines or cytosine analogues that base pair with guanosine.

7. The method according to claim 6, wherein the TSO includes a 3′

portion comprising a plurality of guanosines or guanosine analogues that base pair with cytosine.

8. The method according to claim 7, wherein the guanosines are ribonucleosides or locked nucleic acid monomers.

9. The method according to claim 5, wherein the CDS or the TSO includes a tag.

10. The method according to claim 5, wherein both the CDS and the TSO include a tag.

11. The method according to claim 10, wherein the CDS and the TSO each include a different tag, such that the tagged cDNA sample comprises a combination of tags.

12. The method according to claim 9, wherein the tag is a nucleotide sequence of 4-20 nucleotides in length.

13. The method according to claim 1, wherein each cDNA sample has a distinct tag or combination of tags.

14. The method according to claim 3, wherein the CDS comprises a 5′

amplification primer sequence (APS) and a 3′

RCS.

15. The method according to claim 14, wherein the 3′

RCS comprises oligo(dT), a gene family-specific sequence, a random sequence or a non-self-complementary semi-random sequence.

16. The method according to claim 14, wherein the TSO includes a 5′

APS.

17. The method according to claim 16, wherein the CDS and the 5′

APS of the TSO is at least 80% identical to the 5′

APS of the CDS.

18. The method according to claim 16, wherein the CDS and the 5′

APS of the TSO is 100% identical to the 5′

APS of the CDS.

19. The method according to claim 1, wherein the cells are lysed to release mRNA.

20. The method according to claim 1, wherein the mRNA is purified following step (i).

21. The method according to claim 1, wherein the synthesis of cDNA from mRNA is stopped before the tagged cDNA samples are pooled.

22. The method according to claim 1, wherein the tagged cDNA samples are purified before amplification of the cDNA.

23. The method according to claim 1, wherein in step (iv) the pooled cDNA samples are amplified by PCR.

26. The method according to claim 1, wherein the method further comprises processing the cDNA library to obtain a library suitable for sequencing.

30. A cDNA library produced by the method of claim 1.

31. A method for analysing gene expression in a plurality of single cells, the method comprising the steps of:

preparing a cDNA library according to the method of claim 1; and

sequencing the cDNA library.

32. The method according to claim 31, wherein sequencing is by shotgun sequencing.

33. The method according to claim 32, wherein the cDNA library is sequenced to obtain at least 10,000, at least 1 million, at least 10 million, at least 100 million, or at least 1 billion reads, wherein a read is a length of continuous nucleic acid obtained by a sequencing reaction.

24. (canceled)

25. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)