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Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq

Abstract

Guanine-rich RNAs can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), and participate in a wide range of biological processes. Here we describe in detail a G4-RNA-specific precipitation (G4RP) protocol, which enables the transcriptomic profiling of G4-RNAs. The G4RP protocol consists of a chemical cross-linking step, followed by affinity capture with a G4-specific probe, BioTASQ. G4RP can be coupled with sequencing to capture a comprehensive global snapshot of folded G4-RNAs. This method can also be used to profile induced changes (i.e., through G4 ligand treatments) within the G4-RNA transcriptome. The entire protocol can be completed in 1–2 weeks and can be scaled up or down depending on the specific experimental goals. In addition to the protocol details, we also provide here a guide for optimization in different laboratory setups.

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Fig. 1: Overview of G4RP.
Fig. 2: Expected results from G4RP.
Fig. 3: Expected results from G4RP-seq.

Data availability

All relevant data are available upon request. The G4RP-qPCR dataset (Fig. 2) is publicly available on FigShare (https://doi.org/10.6084/m9.figshare.16684870). The raw sequencing data used in the examples shown in Fig. 3 in this protocol are publicly available and accessible at GSE112898. These raw data are part of the dataset used in ref. 16.

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Acknowledgements

This work is supported by the Research Reinvestment Funds from the University of British Columbia and Agence Nationale de la Recherche (ANR-17-CE17-0010-01). P. Lejault is warmly acknowledged for her critical analysis of the G4RP protocol and of the manuscript.

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Authors

Contributions

S.Y.Y., D.M. and J.M.Y.W. designed the experiments. S.Y.Y. and J.M.Y.W. wrote the manuscript with input from D.M.

Corresponding author

Correspondence to Judy M. Y. Wong.

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Competing interests

D.M. works with Merck-Sigma-Millipore on the commercialization of BioTASQ, the key G4-probe used in this protocol. The remaining authors declare no competing interests.

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Nature Protocols thanks Catherine Merrick and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Yang, S. et al. Nat. Commun. 9, 4730 (2018): https://doi.org/10.1038/s41467-018-07224-8

Renard, I. et al. Nucleic Acids Res. 47, 5502–5510 (2019): https://doi.org/10.1093/nar/gkz215

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Yang, S.Y., Monchaud, D. & Wong, J.M.Y. Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq. Nat Protoc 17, 870–889 (2022). https://doi.org/10.1038/s41596-021-00671-6

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