Protocol abstract


Nature Protocols 5, 516 - 535 (2010)
Published online: 25 February 2010 | doi:10.1038/nprot.2009.236

Subject Categories: Genetic analysis | Genomics and proteomics

RNA-Seq analysis to capture the transcriptome landscape of a single cell

Fuchou Tang1, Catalin Barbacioru2, Ellen Nordman2, Bin Li2, Nanlan Xu2, Vladimir I Bashkirov2, Kaiqin Lao2 & M Azim Surani1


We describe here a protocol for digital transcriptome analysis in a single mouse oocyte and blastomere using a deep-sequencing approach. In this method, individual cells are isolated and transferred into lysate buffer by mouth pipette, followed by reverse transcription carried out directly on the whole cell lysate. Free primers are removed by exonuclease I and a poly(A) tail is added to the 3' end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. Single-cell cDNAs are then amplified by 20 + 9 cycles of PCR. The resulting 100–200 ng of amplified cDNAs are used to construct a sequencing library, which can be used for deep sequencing using the SOLiD system. Compared with cDNA microarray techniques, our assay can capture up to 75% more genes expressed in early embryos. This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d.

Top
  1. Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, UK.
  2. Genetic Systems, Applied Biosystems, part of Life Technologies, Foster City, California, USA.

Correspondence to: M Azim Surani1 e-mail: a.surani@gurdon.cam.ac.uk

Correspondence to: Kaiqin Lao2 e-mail: kai.lao@lifetech.com