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mRNA-Seq whole-transcriptome analysis of a single cell

Nature Methods volume 6, pages 377382 (2009) | Download Citation


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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.

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Change history

  • 19 April 2009

    In the version of this article initially published online, Figure 2d was a duplicate of Figure 2c. The error has been corrected for the print, PDF and HTML versions of this article.


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We thank C. Lee for excellent technical help. The work was supported by grants from the Wellcome Trust to M.A.S.

Author information

Author notes

    • Fuchou Tang
    •  & Catalin Barbacioru

    These authors contributed equally to this work.


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

    • Fuchou Tang
    •  & M Azim Surani
  2. Molecular Cell Biology Division, Applied Biosystems, Foster City, California, USA.

    • Catalin Barbacioru
    • , Yangzhou Wang
    • , Ellen Nordman
    • , Clarence Lee
    • , Nanlan Xu
    • , Xiaohui Wang
    • , John Bodeau
    • , Brian B Tuch
    • , Asim Siddiqui
    •  & Kaiqin Lao


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K.L. designed the project. C.B., B.B.T., A.S., X.W. and K.L. contributed to data analysis, F.T. and M.A.S. contributed to the cDNA sample preparation, E.N., N.X. and Y.W. constructed libraries, C.L. and J.B. contributed to the library sequencing, F.T., E.N. and K.L. contributed to experimental validation, F.T., K.L. and M.A.S. wrote manuscript.

Competing interests

C.B., Y.W., E.N., C.L., N.X., X.W., J.B., B.B.T., A.S., and K.L. are currently employees of Applied Biosystems.

Corresponding authors

Correspondence to Kaiqin Lao or M Azim Surani.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–12

Text files

  1. 1.

    Supplementary Table 1

    The number of reads for RefSeq transcripts of single wild-type, Dicer1−/−, Ago2−/− mature oocytes, and blastomeres of four-cell stage embryos.

  2. 2.

    Supplementary Table 2

    The Ct values of 11 genes which were detected by microarrays but not detected by our mRNA-Seq.

  3. 3.

    Supplementary Table 3

    The Ct values of 380 early embryo marker genes of single wild-type, Dicer1−/−, Ago2−/− mature oocytes, and a single blastomere at the four-cell embryo stage. The corresponding number of reads for these genes by mRNA-Seq is also listed.

  4. 4.

    Supplementary Table 4

    The number of reads for potential novel exon-exon junctions of RefSeq transcripts of single wild-type, Dicer1−/−, Ago2−/− mature oocytes, and blastomeres of four-cell stage embryos.

  5. 5.

    Supplementary Table 5

    The Ct values of 8 potential novel junctions for blastomeres of four-cell stage embryos.

  6. 6.

    Supplementary Table 6

    The number of reads for RefSeq transcripts with multiple known transcript isoforms of single wild-type, Dicer1−/−, Ago2−/− mature oocytes, and a blastomere at the four-cell embryo stage.

  7. 7.

    Supplementary Table 7

    The Ct values of exon-22, exon-23, and exon-24 specific real time PCR assays for the Dicer1 gene to confirm the deletion of exon 23 in the Dicer1−/− oocyte.

  8. 8.

    Supplementary Table 8

    The fold changes, p-values, and FDR values of expressed genes in Dicer1−/− and Ago2−/− mature oocytes compared with wild-type controls based on global quanta normalized reads of single cell mRNA-Seq. We first quantile normalized the mRNA-Seq reads, then, used the Poisson model for the counts of reads for each transcript10 and a goodness-of-fit test to identify differentially expressed genes between samples, controlling false discovery rate (FDR) at a 5% level.

Excel files

  1. 1.

    Supplementary Table 9

    Sequences of the primers used for single cell mRNA-Seq. P1 and P2 Adaptors are same as the SOLiD P1 and P2 Adaptors for library preparation; Library PCR Primer 1 and 2 – SOLiD library amplification primer sets. Amine modification at the 5' end prevents the ligation of the 5' end fragments of the double-stranded cDNA (after the shearing) to the SOLiD library adaptors, thereby eliminating end bias during sequencing.

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