Brief Communication | Published:

Smart-seq2 for sensitive full-length transcriptome profiling in single cells

Nature Methods volume 10, pages 10961098 (2013) | Download Citation

Abstract

Single-cell gene expression analyses hold promise for characterizing cellular heterogeneity, but current methods compromise on either the coverage, the sensitivity or the throughput. Here, we introduce Smart-seq2 with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells. Smart-seq2 transcriptome libraries have improved detection, coverage, bias and accuracy compared to Smart-seq libraries and are generated with off-the-shelf reagents at lower cost.

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Gene Expression Omnibus

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Gene Expression Omnibus

Sequence Read Archive

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Acknowledgements

We thank D. Topcic and N. Volakakis for providing cells, D. Ramsköld for contributing code and the members of the Sandberg laboratory for constructive comments on the work. This work was supported by European Research Council Starting Grant 243066 (R.S.), Swedish Foundation for Strategic Research FFL4 (R.S.) and Swedish Research Council grants 2008-4562 (R.S.) and 2010-6844 (Å.K.B.).

Author information

Affiliations

  1. Ludwig Institute for Cancer Research, Stockholm, Sweden.

    • Simone Picelli
    • , Åsa K Björklund
    • , Omid R Faridani
    • , Sven Sagasser
    • , Gösta Winberg
    •  & Rickard Sandberg
  2. Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

    • Åsa K Björklund
    • , Sven Sagasser
    • , Gösta Winberg
    •  & Rickard Sandberg

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Contributions

S.P. developed the protocol, picked cells, generated cDNA and sequencing libraries, and wrote the manuscript; Å.K.B. performed computational analyses, prepared figures and wrote the manuscript; O.R.F. conceived and designed LNA-based oligos; S.S. picked cells; G.W. contributed to protocol development. R.S. designed the study and wrote the manuscript.

Competing interests

The Ludwig Institute for Cancer Research has submitted a patent application on LNA-based template switching.

Corresponding author

Correspondence to Rickard Sandberg.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–11

Excel files

  1. 1.

    Supplementary Table 1

    Listing and analyses of cDNA yield and length from purified RNA

  2. 2.

    Supplementary Table 2

    Listing of all TSO sequences tested

  3. 3.

    Supplementary Table 3

    Tables of cDNA library yield and length starting with 262 individual human and mouse cells

  4. 4.

    Supplementary Table 4

    Detailing variants of the Smartseq2 protocol

  5. 5.

    Supplementary Table 5

    Cost calculations for cells prepared using SMARTer or Smart-seq2

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DOI

https://doi.org/10.1038/nmeth.2639

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