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

Nature Protocols volume 9, pages 171–181 (2014)Cite this article

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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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Figure 1: Flowchart for Smart-seq2 library preparation.
Figure 2: Bioanalyzer electropherograms of pre-amplified cDNA libraries.
Figure 3: Bioanalyzer electropherogram of a sequencing library after tagmentation.

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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.).

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Authors and 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

Authors
  1. Simone Picelli
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  2. Omid R Faridani
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  4. Gösta Winberg
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  5. Sven Sagasser
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  6. 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.

Corresponding author

Correspondence to Rickard Sandberg.

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Competing interests

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

Supplementary information

Supplementary Table 1

Read statistics for Smart-seq2 single-cell transcriptome libraries from HEK293T and DG-75 cells. (XLSX 51 kb)

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Picelli, S., Faridani, O., Björklund, Å. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc 9, 171–181 (2014). https://doi.org/10.1038/nprot.2014.006

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