ICLIP PROTOCOL TIME SOFTWARE
Enabling amplification of truncated cDNAs led to identification of the sites of RNA-protein interactions at high resolution by analysing the starting position of truncated cDNAs, as well as their precise quantification using UMIs with software called " iCount". iCLIP also added a random sequence (unique molecular identifier, UMI) along with experimental barcodes to the primer used for reverse transcription, thereby barcoding unique cDNAs to minimise any errors or quantitative biases of PCR, and thus improving the quantification of binding events. The RNA is then reverse transcribed, causing most cDNAs to truncate at the crosslink site, and the key innovation and unique feature in the development of iCLIP was to enable such truncated cDNAs to be PCR amplified and sequenced using a next-generation sequencing platform. The labelled protein-RNA complexes are then visualised for quality control, excised from nitrocellulose, and treated with proteinase to release the RNA, leaving only a few amino acids at the crosslink site of the RNA. As with all CLIP methods, iCLIP allows for a very stringent purification of the linked protein-RNA complexes by stringent washing during immunoprecipitation followed by SDS-PAGE and transfer to nitrocellulose. This crosslinking step has generally less background than standard RNA immunoprecipitation (RIP) protocols, because the covalent bond formed by UV light allows RNA to be fragmented, followed by stringent purification, and this also enables CLIP to identify the positions of protein-RNA interactions. ICLIP (individual-nucleotide resolution CrossLinking and ImmunoPrecipitation) is a variant of the original CLIP method used for identifying protein-RNA interactions, which uses UV light to covalently bind proteins and RNA molecules to identify RNA binding sites of proteins. Not to be confused with Intramembrane protease, sometimes abbreviated I-CLiP.
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