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Full-length RNA-seq from single cells using Smart-seq2

Nature Protocols volume 9, pages 171181 (2014) | Download Citation

Abstract

Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes 2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1–3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA) RNA.

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Acknowledgements

This work was supported by the European Research Council Starting grant no. 243066 (R.S.), the Swedish Foundation for Strategic Research FFL4 (R.S.), the Åke Wiberg Foundation (R.S.) and the Swedish Research Council grant nos. 2008-4562 (R.S.) and 2010-6844 (Å.K.B.).

Author information

Affiliations

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

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

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

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Contributions

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

Competing interests

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

Corresponding author

Correspondence to Rickard Sandberg.

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1

    Read statistics for Smart-seq2 single-cell transcriptome libraries from HEK293T and DG-75 cells.

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DOI

https://doi.org/10.1038/nprot.2014.006

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