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Genome-wide mapping of RNA structure using nuclease digestion and high-throughput sequencing

Abstract

RNA structure is important for RNA function and regulation, and there is growing interest in determining the RNA structure of many transcripts. Here we provide a detailed protocol for the parallel analysis of RNA structure (PARS) for probing RNA secondary structures genome-wide. In this method, enzymatic footprinting is coupled to high-throughput sequencing to provide secondary structure data for thousands of RNAs simultaneously. The entire experimental protocol takes 5 d to complete, and sequencing and data analysis take an additional 6–8 d. PARS was developed using the yeast genome as proof of principle, but its approach should be applicable to probing RNA structures from different transcriptomes and structural dynamics under diverse solution conditions.

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Figure 1: Detailed experimental schematic of PARS.
Figure 2: Detailed analysis pipeline of PARS.
Figure 3: PARS correctly recapitulates results of RNA footprinting for the P9-9.2 domain of the Tetrahymena ribozyme10.
Figure 4: Probing melting dynamics of the RNA structure across different temperatures.

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Acknowledgements

This work was supported by National Institutes of Health (R01-HG004361). Y.W. is funded by the Agency of Science, Technology and Research of Singapore. H.Y.C. is an Early Career Scientist of the Howard Hughes Medical Institute.

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Contributions

Y.W. and H.Y.C. developed the protocol and designed the experiments; Y.W. performed the experiments; K.Q. analyzed the data; Z.O. developed the SeqFold pipeline; Y.W. and H.Y.C. wrote the paper with contributions from all authors.

Corresponding author

Correspondence to Howard Y Chang.

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Competing interests

Y.W. and H.Y.C. are named as inventors on a patent application filed on the PARS method by Weizmann Institute and Stanford University.

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Wan, Y., Qu, K., Ouyang, Z. et al. Genome-wide mapping of RNA structure using nuclease digestion and high-throughput sequencing. Nat Protoc 8, 849–869 (2013). https://doi.org/10.1038/nprot.2013.045

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