Transcriptome-wide identification of RNA-binding protein binding sites using seCLIP-seq.

Autor: Blue SM; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA., Yee BA; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA., Pratt GA; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA., Mueller JR; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA., Park SS; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA., Shishkin AA; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA.; Eclipse Bioinnovations, San Diego, CA, USA., Starner AC; Verna & Marrs McLean Department of Biochemistry & Molecular Biology and Therapeutic Innovation Center, Baylor College of Medicine, Houston, TX, USA., Van Nostrand EL; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA.; Verna & Marrs McLean Department of Biochemistry & Molecular Biology and Therapeutic Innovation Center, Baylor College of Medicine, Houston, TX, USA., Yeo GW; Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA. geneyeo@ucsd.edu.
Jazyk: angličtina
Zdroj: Nature protocols [Nat Protoc] 2022 May; Vol. 17 (5), pp. 1223-1265. Date of Electronic Publication: 2022 Mar 23.
DOI: 10.1038/s41596-022-00680-z
Abstrakt: 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.
(© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)
Databáze: MEDLINE
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