Journal
NATURE PROTOCOLS
Volume 9, Issue 2, Pages 263-293Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2014.012
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Funding
- Jane Coffin Childs Fund for Medical Research
- US National Institutes of Health (NIH) [K99/R00GM95713]
- Simons Foundation [297990]
- Simons Foundation
- Howard Hughes Medical Institute
- [NIH NS081706]
- [NIH NS34389]
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The identification of sites where RNA-binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV cross-linking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from in vivo cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high-throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide maps of RNA binding with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. The application of CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of cross-link-induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes similar to 8 d to prepare RNA for sequencing. Established pipelines for data analysis, including those for CIMS, take 3-4 d.
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