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Remove media and add ice-cold PBS to cells (eg use cells grown in a 10 cm plate for three experiments and add 6 ml PBS).ġ.2. UV cross linking of tissue culture cellsġ.1. Protease inhibitors (add fresh each time)ġ/500 RNase I dilutions for library preparation 1/50 RNase I dilutions to control for antibodyĤ μL 5x PNK pH 6.5 buffer Ĥ μL 4x ligation buffer Ġ.25 μL Superscript III reverse transcriptase “iCLIP: Protein–RNA interactions at nucleotide resolution.” “iCLIP -Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution.”įurther adapted from Huppertz et al. Here is a summary of the iCLIP protocol adapted from Konig et al. Unlike RIP, CLIP provides information about the actual protein binding site on the RNA.ĭifferent types of CLIP exist, high-throughput sequencing-CLIP (HITS-CLIP), photoactivatable-ribonucleoside enhanced CLIP (PAR-CLIP), and individual CLIP (iCLIP). This covalent bond is irreversible, allowing stringent purification conditions. Cell Rep.Interest in RNA-protein interactions is booming as we begin to appreciate the role of RNA, not just in well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs.ĬLIP is an antibody-based technique used to study RNA-protein interactions related to RNA immunoprecipitation (RIP) but differs from RIP in the use of UV radiation to cross-link RNA binding proteins to the RNA that they are bound to. Elucidating MicroRNA Regulatory Networks Using Transcriptional, Post-transcriptional, and Histone Modification Measurements. Radioisotopes required for visualization of UV-crosslinked protein-RNA complexes only label 5' ends (Zarnegar et al., 2016).Other versions: HITS-CLIP, PAR-CLIP, eCLIP, irCLIP Advantages: 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).
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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).