Abstract
We present a detailed protocol for the experimental identification of miRNA-target RNA interaction sites using cross-linking, ligation, and sequencing of hybrids (CLASH). The basis of the technique is the purification of UV-stabilized Argonaute (AGO)–RNA complexes assembled in living cells, with subsequent ligation of AGO-associated RNA-RNA duplexes to form chimeric RNAs. Following cDNA synthesis, DNA library preparation and high-throughput sequencing, interacting RNA molecules are unambiguously identified as chimeric reads in bioinformatic analysis of sequencing data. CLASH potentially recovers any RNA duplex that is bound by RNA-binding protein, so modified approaches would be suitable for the identification of many other inter- and intramolecular RNA-RNA interactions. Since CLASH analysis is independent of bioinformatic predictions it allows the identification and analysis of RNA targeting rules in an unbiased way.
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Helwak, A., Tollervey, D. (2016). Identification of miRNA-Target RNA Interactions Using CLASH. In: Dassi, E. (eds) Post-Transcriptional Gene Regulation. Methods in Molecular Biology, vol 1358. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3067-8_14
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DOI: https://doi.org/10.1007/978-1-4939-3067-8_14
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3066-1
Online ISBN: 978-1-4939-3067-8
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