RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.
Keywords: High-throughput sequencing; Post-transcriptional regulation; Protein–RNA interaction; RNA; RNA-binding protein; UV crosslinking and immunoprecipitation (CLIP); iCLIP.
Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.