Abstract
Discovery of interaction sites between RNA-binding proteins (RBPs) and their RNA targets plays a critical role in enabling our understanding of how these RBPs control RNA processing and regulation. Cross-linking and immunoprecipitation (CLIP) provides a generalizable, transcriptome-wide method by which RBP/RNA complexes are purified and sequenced to identify sites of intermolecular contact. By simplifying technical challenges in prior CLIP methods and incorporating the generation of and quantitative comparison against size-matched input controls, the single-end enhanced CLIP (seCLIP) protocol allows for the profiling of these interactions with high resolution, efficiency and scalability. Here, we present a step-by-step guide to the seCLIP method, detailing critical steps and offering insights regarding troubleshooting and expected results while carrying out the ~4-d protocol. Furthermore, we describe a comprehensive bioinformatics pipeline that offers users the tools necessary to process two replicate datasets and identify reproducible and significant peaks for an RBP of interest in ~2 d.
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Code availability
All code is made freely and publicly available under the BSD-3 license. Custom scripts and workflow definition files described in this paper may be found at https://doi.org/10.5281/zenodo.507659149. Up-to-date versions may be found on GitHub at https://github.com/yeolab/eclip.
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Acknowledgements
The authors thank Yeo laboratory member K. Rothamel for critical reading of the manuscript. This work was supported by grants from the US National Institutes of Health (HG009889 and HG004659 to G.W.Y. and HG009530 to E.L.V.N.).
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S.M.B. wrote the sections of the manuscript pertaining to the experimental protocol and contributed to the development of the experimental methods. B.A.Y. wrote the sections of the manuscript pertaining to the bioinformatics methods. G.A.P. contributed to the development of bioinformatics methods. J.R.M. and S.S.P. contributed to developing the experimental methods and produced the data in Fig. 3. A.A.S. contributed to the development of experimental methods. A.C.S. produced the data presented in Supplementary Figure 2. E.L.V.N. contributed to the development of all experimental and bioinformatics methods described and directed writing of the manuscript. G.W.Y. directed the development of all experimental and bioinformatics methods described and the writing of the manuscript.
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G.W.Y. is co-founder, member of the Board of Directors, on the Science Advisory Board, an equity holder and a paid consultant for Locanabio and Eclipse BioInnovations. G.W.Y. is a visiting professor at the National University of Singapore. G.W.Y.’s interest(s) have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. A.A.S. is co-founder and Research & Development Director for Eclipse Bioinnovations. E.L.V.N. is co-founder, member of the Board of Directors, on the Science Advisory Board, an equity holder and a paid consultant for Eclipse BioInnovations. E.L.V.N.’s interest(s) have been reviewed and approved by Baylor College of Medicine in accordance with its conflict-of-interest policies. The authors declare no other competing interests.
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Key references using this protocol
Van Nostrand, E. et al. Nat. Methods 13, 508–514 (2016): https://doi.org/10.1038/nmeth.3810
Van Nostrand, E. et al. Nature 583, 711–719 (2020): https://doi.org/10.1038/s41586-020-2077-3
Van Nostrand, E. et al. Methods Mol. Biol. 1648, 177–200 (2017): https://doi.org/10.1007/978-1-4939-7204-3_14
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Blue, S.M., Yee, B.A., Pratt, G.A. et al. Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq. Nat Protoc 17, 1223–1265 (2022). https://doi.org/10.1038/s41596-022-00680-z
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DOI: https://doi.org/10.1038/s41596-022-00680-z
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