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Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE

Nature Protocols volume 11, pages 273290 (2016) | Download Citation

Abstract

icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3–modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in 5 d, and the sequencing and bioinformatics data analysis take an additional 4–5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models.

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Acknowledgements

We thank E. Kool and Kool laboratory members for synthesis of NAI-N3. This study was supported by National Institutes of Health grant nos. NIH R01-HG004361 and NIH P50-HG007735, and by the Howard Hughes Medical Institute (H.Y.C.).

Author information

Author notes

    • Ryan A Flynn
    •  & Qiangfeng Cliff Zhang

    These authors contributed equally to this work.

Affiliations

  1. Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, California, USA.

    • Ryan A Flynn
    • , Qiangfeng Cliff Zhang
    • , Robert C Spitale
    • , Byron Lee
    • , Maxwell R Mumbach
    •  & Howard Y Chang

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Contributions

R.A.F., Q.C.Z., R.C.S. and H.Y.C. designed the experimental and computational strategy. R.A.F., R.C.S., B.L. and M.R.M. optimized experimental conditions. Q.C.Z. optimized computational parameters. R.A.F., Q.C.Z. and H.Y.C. wrote the manuscript with input from all authors.

Competing interests

H.Y.C. is an inventor on a patent for in vivo SHAPE reagents. H.Y.C. is a founder of Epinomics and a member of the Scientific Advisory Board of RaNA Therapeutics.

Corresponding author

Correspondence to Howard Y Chang.

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DOI

https://doi.org/10.1038/nprot.2016.011

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