Journal Article

Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)

Van Nostrand, Eric L, Gelboin-Burkhart, Chelsea, Elkins, Keri, Pratt, Gabriel A, Stanton, Rebecca, Surka, Christine, Yeo, Gene W, Shishkin, Alexander A, Fang, Mark Y, Guttman, Mitchell, Nguyen, Thai B, Sundararaman, Balaji, Rigo, Frank, Blue, Steven M

2016

Overview

Enhanced CLIP yields complex libraries of RNA components of ribonucleoprotein complexes and maintains single-nucleotide resolution of binding sites. eCLIP enables large scale profiling, as demonstrated with the binding profiles of 73 RBPs in two human cancer cell lines. As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org ), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 38/77 / 38/91 / 45 / 45/90 / 631/1647/2217 / 631/1647/514/1949 / 631/337/1645 / 631/61/212/2019 / 82/1 / Amplification / Analysis / Binding proteins / Binding Sites / Bioinformatics / Biological Microscopy / Biological Techniques / Biomedical Engineering/Biotechnology / Complexity / Cross-Linking Reagents - chemistry / Crosslinking / Failure rates / Gene amplification / Gene Expression Profiling - methods / Genetic research / Hep G2 Cells / Humans / Immunoassay / Immunoglobulin G / Immunoprecipitation / Immunoprecipitation - methods / Innovations / K562 Cells / Libraries / Life Sciences / Methods / Nucleotides / Photochemical Processes / Physiology / Properties / Protein binding / Proteins / Proteomics / Ribonucleic acid / Ribonucleoproteins / RNA / RNA-binding protein / RNA-Binding Proteins - genetics / RNA-protein interactions / Robustness / Transcriptome / Transcriptomes / Tumor cell lines / Ultraviolet Rays