Language
English
Publication Date
5-1-2022
Journal
Nature Protocols
DOI
10.1038/s41596-022-00680-z
PMID
35322209
PMCID
PMC11134598
PubMedCentral® Posted Date
5-29-2024
PubMedCentral® Full Text Version
Author MSS
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.
Keywords
Binding Sites, High-Throughput Nucleotide Sequencing, Immunoprecipitation, Protein Binding, RNA, RNA-Binding Proteins, Transcriptome
Published Open-Access
yes
Recommended Citation
Blue, Steven M; Yee, Brian A; Pratt, Gabriel A; et al., "Transcriptome-Wide Identification of RNA-Binding Protein Binding Sites Using seCLIP-Seq" (2022). Faculty and Staff Publications. 278.
https://digitalcommons.library.tmc.edu/baylor_docs/278
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Biochemistry, Biophysics, and Structural Biology Commons, Biology Commons, Genetic Phenomena Commons, Genetics Commons, Genomics Commons, Medical Genetics Commons, Medical Specialties Commons