期刊
NATURE METHODS
卷 13, 期 6, 页码 508-+出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.3810
关键词
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资金
- US National Institutes of Health [HG004659, U54HG007005, NS075449]
- US National Institutes of Health Director's Early Independence Award [DP50D012190]
- California Institute of Technology
- National Science Foundation
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 similar to 1,000-fold, decreasing discarded PCR duplicate reads by similar to 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.
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