Transcriptome-Wide Identification of RNA-Binding Protein Binding Sites Using seCLIP-Seq

Authors

Steven M Blue
Brian A Yee
Gabriel A Pratt
Jasmine R Mueller
Samuel S Park
Alexander A Shishkin
Anne C Starner
Eric L Van Nostrand
Gene W Yeo

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

Published Open-Access

no

Keywords

Binding Sites, High-Throughput Nucleotide Sequencing, Immunoprecipitation, Protein Binding, RNA, RNA-Binding Proteins, Transcriptome

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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