![]() ![]() 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.Overall, the methodological advances in iCLIP2 allow efficient library generation and thereby promote the versatile and flexible application of this important technology.ĬLIP High-throughput sequencing Protein-RNA interaction RNA-binding protein UV crosslinking iCLIP.Ĭopyright © 2019 The Authors. Our advances significantly increase the complexity of the iCLIP2 libraries, resulting in a more comprehensive representation of RBP binding sites. The full procedure can be completed within four days. The new protocol comprises separate adapter ligations, two cDNA amplification steps and bead-based size selection. Here, we present the new iCLIP2 protocol that allows to obtain high-quality iCLIP libraries in a fast and efficient manner. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) is a state-of-the-art technology to map the RNA interaction sites of an RNA-binding protein (RBP) across the transcriptome. ![]()
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