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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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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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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Supplementary Text and Figures
Supplementary Figures 1–15 and Supplementary Note 1 (PDF 3157 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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DOI: https://doi.org/10.1038/nmeth.4582
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