title: Mapping the miRNA interactome by cross-linking ligation and sequencing of hybrids (CLASH).
abstract: RNA-RNA interactions have critical roles in many cellular processes, but studying them is difficult and laborious. Here we describe an experimental procedure, termed cross-linking ligation and sequencing of hybrids (CLASH), which allows high-throughput identification of sites of RNA-RNA interaction. During CLASH, a tagged bait protein is UV-cross-linked in cell cultures to stabilize RNA interactions, and it is purified under denaturing conditions. RNAs associated with the bait protein are partially truncated, and the ends of RNA duplexes are ligated together. After linker addition, cDNA library preparation and high-throughput sequencing, the ligated duplexes give rise to chimeric cDNAs, which unambiguously identify RNA-RNA interaction sites independent of bioinformatic predictions. This protocol is optimized for studying miRNA targets bound by Argonaute (AGO) proteins, but it should be easily adapted for other RNA-binding proteins and classes of RNA. The protocol requires ∼5 d to complete, excluding the time required for high-throughput sequencing and bioinformatic analyses.
authors.affiliation: Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Edinburgh, UK.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Helwak
authors.affiliation: Wellcome Trust Centre for Cell Biology, The University of Edinburgh, Edinburgh, UK.
authors.forename: David
authors.identifier: 0000000328942772
authors.initials: D
authors.lastname: Tollervey
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods; D019295:Computational Biology / Q000379:methods; D003432:Cross-Linking Reagents / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D060565:Argonaute Proteins; D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D035683:MicroRNAs