title: Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP).
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Eric L
authors.initials: EL
authors.lastname: Van Nostrand
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.|Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, California, USA.
authors.forename: Gabriel A
authors.initials: GA
authors.lastname: Pratt
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Shishkin
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Chelsea
authors.initials: C
authors.lastname: Gelboin-Burkhart
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Mark Y
authors.initials: MY
authors.lastname: Fang
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Balaji
authors.initials: B
authors.lastname: Sundararaman
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Blue
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Thai B
authors.initials: TB
authors.lastname: Nguyen
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Surka
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Keri
authors.initials: K
authors.lastname: Elkins
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Stanton
authors.affiliation: Ionis Pharmaceuticals, Carlsbad, California, USA.
authors.forename: Frank
authors.initials: F
authors.lastname: Rigo
authors.affiliation: Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
authors.forename: Mitchell
authors.initials: M
authors.lastname: Guttman
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.|Stem Cell Program, University of California at San Diego, La Jolla, California, USA.|Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.|Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, California, USA.|Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.|Molecular Engineering Laboratory, A*STAR, Singapore.
authors.forename: Gene W
authors.initials: GW
authors.lastname: Yeo
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D056945:Hep G2 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D020014:K562 Cells; D055668:Photochemical Processes; D016601:RNA-Binding Proteins / Q000235:genetics*; D059467:Transcriptome; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D016601:RNA-Binding Proteins