GENE EXPRESSION ANALYSIS IN SINGLE CELLS
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.
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.
24 . (canceled)
25 . (canceled)
26 . The method according to claim 1 , wherein the method further comprises processing the cDNA library to obtain a library suitable for sequencing.
27 . (canceled)
28 . (canceled)
29 . (canceled)
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.