Other versions: HITS-CLIP, PAR-CLIP, eCLIP, irCLIP Advantages: Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements. The PSI-U1 snRNP interaction regulates male mating behavior in Drosophila. Global analysis of CPSF2-mediated alternative splicing: Integration of global iCLIP and transcriptome profiling data. IGF2BP3 Modulates the Interaction of Invasion-Associated Transcripts with RISC. M., Sterne-Weiler T., Jahanbani F., Coyne D.
Profiling the Binding Sites of RNA-Binding Proteins with Nucleotide Resolution Using iCLIP. High-throughput characterization of protein-RNA interactions. Illumina Library prep and Array Kit SelectorĬook K. Circular ligation can be inefficient (Van Nostrand et al., 2016).Radioisotopes required for visualization of UV-crosslinked protein-RNA complexes only label 5′ ends (Zarnegar et al., 2016).Artifacts may be introduced in the circularization step.Nonlinear PCR amplification can lead to biases affecting reproducibility.Antibodies not specific to target will precipitate nonspecific complexes.Amplification allows the detection of rare events.Nucleotide resolution of protein-binding sites.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 (Van Nostrand et al., 2016). These circularized fragments are linearized and PCR-amplified. Upon RT, cDNA truncates at the binding site and is circularized. The complexes are treated with proteinase K, as the protein crosslinked at the binding site remains undigested. 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 (Konig et al., 2010).
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