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

Abstract

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.

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Figure 1: Workflow for SOLiD System Express library preparation for fragment libraries and multiplexed fragment libraries.
Figure 2: cDNA library fragment QC.
Figure 3: PCR amplified single-cell cDNA libraries.
Figure 4: Standard curve of cDNAs following qPCR quantification.
Figure 5: Single mouse embryonic stem (ES) cells in a PBS–BSA drop.
Figure 6: GAPDH expression in mouse embryonic stem (ES) cells measured by real-time PCR.
Figure 7: Assessing accuracy and reproducibility of the single-cell RNA-Seq method.
Figure 8: Coverage plots of RNA-Seq reads from a single wild-type mature oocyte.

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Acknowledgements

We thank John Bodeau, Clarence Lee, Yangzhou Wang, Umberto Ulmanella, Karen Li, Cinna Monighetti and Swati Rande for their generous help.

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Authors

Contributions

K.L. designed the project; F.T. and E.N. carried out the experiments; C.B. analyzed the data; B.L., V.I.B. and N.X. improved the protocols; F.T., E.N., K.L. and M.A.S. wrote the manuscript with contributions from all the authors.

Corresponding authors

Correspondence to Kaiqin Lao or M Azim Surani.

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Competing interests

C.B., E.N., B.L., N.X., V.I.B. and K.L. are currently employees of Applied Biosystems (part of Life Technologies).

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Tang, F., Barbacioru, C., Nordman, E. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nat Protoc 5, 516–535 (2010). https://doi.org/10.1038/nprot.2009.236

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