![]() Other versions: HITS-CLIP, PAR-CLIP, eCLIP, irCLIP. iCLIP: Individual-nucleotide resolution CLIP is a refinement of CLIP that allows single-nucleotide resolution of RBP binding sites. A partial RNase digestion is then employed to restrict the RNA fragment lengths to a defined range. 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. In order to obtain a snapshot of the in vivo RNA binding pattern of an RBP, the iCLIP experiment initiates with the UV irradiation of living cells to covalently crosslink immediate contacts between proteins and nucleic acids (Fig. 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. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1) 14. Here, we developed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. B) Comparison of the iCLIP2 workflow to iCLIP 13, eCLIP 10 and irCLIP 11. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein. iCLIP maps protein-RNA interactions, in a process similar to HITS-CLIP and PAR-CLIP. In iCLIP, specific crosslinked RNA-protein complexes are immunoprecipitated. A) Schematic representation of the different steps of the iCLIP2 workflow. The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. 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. ![]()
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