![]() Other versions: HITS-CLIP, PAR-CLIP, eCLIP, irCLIP. Deep sequencing of these amplified fragments provides nucleotide resolution of the protein-binding site eCLIP is an improvement over iCLIP that avoids circularizing the cDNA to reduce artifacts. The main difference with the original CLIP printers. Carbon’s latest resin 3D-printers printers use a 4K DLP printer with 385nm wavelength, similar as other printers like Asiga, Genera3D and Origin (last are both funded by Stratasys). These circularized fragments are linearized and PCR-amplified. iCLIP is a vat polymerization method using UV-light to cure the resin similar to Carbon’s CLIP technology. Upon RT, cDNA truncates at the binding site and is circularized. In the past decade, leaps forward in biochemistry and high throughput sequencing methods have enabled mapping of RNA modifications across all RNA species. The complexes are treated with proteinase K, as the protein crosslinked at the binding site remains undigested. The iCLIP protocol starts with UV irradiation, which forms covalent bonds at sites of proteinRNA interactions and thereby preserves the in vivo binding pattern. In iCLIP, specific crosslinked RNA-protein complexes are immunoprecipitated. This approach includes additional steps to digest the proteins after crosslinking and to map the crosslink sites with reverse transcriptase. ICLIP maps protein-RNA interactions, in a process similar to HITS-CLIP and PAR-CLIP. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies proteinRNA crosslink sites on a genome-wide scale.
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