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Nat Methods. 2009 May;6(5):377-82. doi: 10.1038/nmeth.1315. Epub 2009 Apr 6.

mRNA-Seq whole-transcriptome analysis of a single cell.

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  • 1Wellcome Trust-Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, UK.

Abstract

Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.

PMID:
19349980
[PubMed - indexed for MEDLINE]
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