Chapter title |
Post-Transcriptional Gene Regulation
|
---|---|
Chapter number | 14 |
Book title |
Post-Transcriptional Gene Regulation
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3067-8_14 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3066-1, 978-1-4939-3067-8
|
Authors |
Helwak, Aleksandra, Tollervey, David, Aleksandra Helwak, David Tollervey |
Editors |
Erik Dassi |
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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