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Title: Enhanced CLIP (eCLIP) enables robust and scalable transcriptome-wide discovery and characterization of RNA binding protein binding sites [array]      
keywords:
Transcriptome or Gene expression
ID:
PRJNA310093
description:
RNA binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNAs. As defects in protein-RNA recognition lead to human disease, UV-crosslinking and immunoprecipitation (CLIP) of ribonuclear complexes followed by deep sequencing (-seq) is critical in constructing protein-RNA maps to expand our understanding of RBP function. However, current CLIP protocols are technically demanding and involve low complexity libraries that yield squandered sequencing of PCR duplicates and high experimental failure rates. To enable truly large-scale implementation of CLIP-seq, we have developed an enhanced CLIP methodology (eCLIP) that features a decrease of ~10 cycles of requisite amplification with a concomitant >60% decrease in discarded PCR duplicate reads, while maintaining the ability to identify RNA binding with single-nucleotide resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP also dramatically improves specificity in discovery of authentic binding sites. To demonstrate that eCLIP enables large-scale and robust profiling of RBPs, 102 eCLIP experiments in biological duplicate for a diverse collection of 74 RBPs in HepG2 and K562 cells were completed (available at https://www.encodeproject.org). We establish that eCLIP is comparable in amplification and sample requirements to ChIP-seq, and enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP experiments and RNA-centric perspectives of RBP activity. Overall design: Microarray profiling of RBFOX2 knockdown by shRNAs in 293T cells
accesstypes:
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landingpage: http://www.ncbi.nlm.nih.gov/bioproject/PRJNA310093
authentication:
none
authorization:
none
ID:
pmid:27018577
name:
Homo sapiens
ncbiID:
ncbitax:9606
abbreviation:
NCBI
homePage: http://www.ncbi.nlm.nih.gov
ID:
SCR:006472
name:
National Center for Biotechnology Information
homePage: http://www.ncbi.nlm.nih.gov/bioproject
ID:
SCR:004801
name:
NCBI BioProject