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Nat Methods. 2016 Jun;13(6):508-14. doi: 10.1038/nmeth.3810. Epub 2016 Mar 28.

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

Author information

1
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, California, USA.
2
Stem Cell Program, University of California at San Diego, La Jolla, California, USA.
3
Institute for Genomic Medicine, University of California at San Diego, La Jolla, California, USA.
4
Bioinformatics and Systems Biology Graduate Program, University of California at San Diego, La Jolla, California, USA.
5
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.
6
Ionis Pharmaceuticals, Carlsbad, California, USA.
7
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
8
Molecular Engineering Laboratory, A*STAR, Singapore.

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.

PMID:
27018577
PMCID:
PMC4887338
DOI:
10.1038/nmeth.3810
[Indexed for MEDLINE]
Free PMC Article

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