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TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding

Nature Methods volume 15, pages 221–225 (2018)Cite this article

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Abstract

RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.

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Figure 1: TimeLapse-seq uses a convertible nucleoside approach to identify new transcripts in a sequencing experiment.
Figure 2: Global analysis of steady-state and transient RNA dynamics using TimeLapse-seq.
Figure 3: TimeLapse-seq reveals differential transcript isoform stability of the ASXL1 transcript.

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Acknowledgements

We thank J. Steitz, A. Schepartz, D. Söll, D. Canzio, and the Simon Lab for insightful comments; and we thank Y. Wang and A. Sexton for assistance and scripts used in mutational analysis of targeted sequencing data. This work was supported by the NIH NIGMS T32GM007223 (J.A.S. and E.E.D.); NSF Graduate Research Fellowship (E.E.D.); NIH New Innovator Award DP2 HD083992-01 (M.D.S.), and a Searle scholarship (M.D.S.).

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Authors and Affiliations

  1. Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, Connecticut, USA

    Jeremy A Schofield, Erin E Duffy, Lea Kiefer, Meaghan C Sullivan & Matthew D Simon

  2. Chemical Biology Institute, Yale University, West Haven, Connecticut, USA

    Jeremy A Schofield, Erin E Duffy, Lea Kiefer, Meaghan C Sullivan & Matthew D Simon

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  1. Jeremy A Schofield
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Contributions

J.A.S. and M.D.S. designed experiments. J.A.S., E.E.D., and L.K. carried out experiments. J.A.S., M.C.S., and M.D.S. performed computational analyses of data. J.A.S. and M.D.S. wrote the manuscript with assistance from all authors.

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Correspondence to Matthew D Simon.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Note 1 (PDF 3157 kb)

Life Sciences Reporting Summary (PDF 159 kb)

Supplementary Protocol

Supplementary Protocol (PDF 433 kb)

Supplementary Table 1

Primers and oligonucleotides used in this study. (XLSX 11 kb)

Supplementary Table 2

Transcript half-lives for individual replicates and combined data for MEF (1h s4 U treatment) and K562 cells (4h s4 U treatment). (XLSX 709 kb)

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Schofield, J., Duffy, E., Kiefer, L. et al. TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding. Nat Methods 15, 221–225 (2018). https://doi.org/10.1038/nmeth.4582

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