Recent studies have indicated that there are over 1500 RBPs, and this number is continuing to expand with additional studies. Recent advancements in next-generation sequencing Despite these known important roles, however, detailed studies to describe the targets of and regulatory mechanisms have largely focused on a small set of RBPs, with the majority of RBPs remaining poorly characterized. Have made it possible to study RBPs and their RNA targets in an unbiased and transcriptome-wide manner. In CLIP, RNA-protein interactions are stabilized via ultravioletĬrosslinking, a desired protein is immunoprecipitated using a factor-specific antibody, and associated RNA is isolated and converted into DNA library Building upon early RNA ImmunoPrecipitation (RIP) approaches that identified protein binding to entire transcripts, CrossLinking and ImmunoPrecipitation (CLIP)Įnabled high-resolution profiling of binding sites. Various modifications of CLIP have since been described, including the use of photoactivatable nucleoside analogs (PNAs) to improve crosslinking efficiency (PAR-CLIP) and computational and experimental methods to identify binding with single-nucleotide resolution. Treatment of UV-crosslinked protein-RNA complexes leaves at least one amino acid covalently crosslinked to its associated ribonucleotide. Step, iCLIP positions this crosslinking site at the start of the sequencing Reverse transcriptase enzymes can create deletions at these positions or, more often, terminate elongation due to the inability to read through this coupling, leading to a substantial fraction of cDNA fragments that terminate at the position of crosslinking. We recently described enhanced CLIP (eCLIP, which leads to high experimental failure rates and high wasted sequencing (often >90% of reads) due to the presence of substantial PCR However, widespread usage of these methods has been limited by the low efficiency of converting RNA molecules into adapter-ligated library Reads to enable identification of binding sites with single-nucleotide resolution. ), which incorporated high-efficiency enzymatic steps to achieve thousand-fold improved library efficiency. The improved efficiency dramatically decreases experimental failure rates and PCR duplication, and enabled quantitative comparison with paired size-matched input to remove common CLIP artifacts. ![]() Here, we describe a detailed protocol for seCLIP, a simplified, single-end version of the eCLIP methodology, as well as assorted notes for critical handling steps.
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