This is followed by partial RNase digestion and an immunoprecipitation with protein-specific antibodies. Cells are irradiated with UV-C light on ice, leading to formation of a covalent bond between protein and RNA. 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. 4 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 Institute of Molecular Biology, Ackermannweg 4, 55128 Mainz, Germany.3 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK Faculty of Medicine, University of Ljubljana, Vrazov Ljubljana, Slovenia.2 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.1 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.Similar methods: iCLIP, irCLIP, HITS-CLIP. Finally, the paired-end cDNA fragments are amplified and sequenced. The resulting cDNA is ligated to single-stranded DNA adapters on the 3' end that contain either an N5 or N10 sequence to serve as unique identifiers against PCR duplicates. The bound protein is removed by proteinase K digestion, and the RNA is reverse-transcribed. Next, the protein-RNA complexes are immunoprecipitated and ligated to an RNA adapter on the 3' end of the target RNA. The hallmark of this method is the ligation of barcoded single-stranded DNA adapters, which reduce amplification bias significantly.įirst, RNA and the protein of interest are UV-crosslinked, followed by cell lysis and RNase I digestion. Method Category: Transcriptome > RNA-Protein Interactionsĭescription: eCLIP maps the binding sites of RBPs on their target RNAs using a modified individual nucleotide resolution CLIP (iCLIP) protocol, improving efficiency and decreasing execution complexity.
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