Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including. T3 - Methods in molecular biology (Clifton, N.J. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP-RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries.ĪB - RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. Global studies using iCLIP-Seq, RNA-Seq and ribosome profiling revealed that many target mRNAs and a wide variety of RNA processes are potentially impacted. N2 - RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. Subsequently, the binding sites have to be visualized. These binding sites can be determined genome-wide through individual nucleotide resolution crosslinking immunoprecipitation (iCLIP). This approach includes additional steps to digest the proteins after. T1 - Individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to determine protein-RNA interactions RNA-binding proteins interact with their target RNAs at specific sites. iCLIP maps protein-RNA interactions, in a process similar to HITS-CLIP and PAR-CLIP.
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