Schematic representation of the iCLIP procedure identifying RNA–protein interactions in intact cells. High-throughput sequencing Post-transcriptional regulation Protein–RNA interaction RNA RNA-binding protein UV crosslinking and immunoprecipitation (CLIP) iCLIP.Ĭopyright © 2013 The Authors. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs. 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. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Electronic address: proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Electronic address: 5 Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
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