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Quantitative single-cell RNA-seq with unique molecular identifiers

Nature Methods volume 11, pages 163166 (2014) | Download Citation

Abstract

Single-cell RNA sequencing (RNA-seq) is a powerful tool to reveal cellular heterogeneity, discover new cell types and characterize tumor microevolution. However, losses in cDNA synthesis and bias in cDNA amplification lead to severe quantitative errors. We show that molecular labels—random sequences that label individual molecules—can nearly eliminate amplification noise, and that microfluidic sample preparation and optimized reagents produce a fivefold improvement in mRNA capture efficiency.

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Acknowledgements

Illumina sequencing experiments were performed by A. Johnsson and A. Juréus (Karolinska Institutet). ES cells were generously provided by the Karolinska Center for Transgene Technologies. Recombinant Tn5 transposase was a generous gift from R. Sandberg (Karolinska Institutet). We are grateful for the advice and support of B. Jones and B. Fowler (Fluidigm). This work was supported by grants from the Swedish Research Council (STARGET) and the European Research Council (261063) to S.L. and from the Swedish Cancer Society, Swedish Research Council and Ragnar Söderberg Foundation to M.K.

Author information

Affiliations

  1. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Saiful Islam
    • , Amit Zeisel
    • , Gioele La Manno
    • , Pawel Zajac
    • , Peter Lönnerberg
    •  & Sten Linnarsson
  2. Department of Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden.

    • Simon Joost
    •  & Maria Kasper

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Contributions

S.L. conceived the study. S.I., A.Z. and P.Z. developed the protocols and performed the RNA-seq experiments. S.J. and M.K. prepared cells. A.Z., G.L.M. and S.I. developed the tagmentation protocol. P.L. wrote the bioinformatics pipeline. P.L., S.L. and A.Z. analyzed data. S.L. and S.I. wrote the manuscript with support from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sten Linnarsson.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–13 and Supplementary Note

Excel files

  1. 1.

    Supplementary Table 1

    List of noisy genes

  2. 2.

    Supplementary Table 2

    List of custom oligonucleotides

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DOI

https://doi.org/10.1038/nmeth.2772