ICLIP

Not to be confused with Intramembrane protease, sometimes abbreviated I-CLiP.

iCLIP (individual-nucleotide resolution Cross-Linking 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. This cross-linking 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. 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. 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.

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. Another transformative innovation of iCLIP was to add a random sequence (unique molecular identifier, UMI) along with experimental barcodes to the primer used for reverse transcription, which is used to barcode unique cDNAs, thus minimising any errors or quantitative biases of PCR [./Https://www.nature.com/articles/s43586-021-00018-1 amplification] and therefore improves the quantification of binding events. With these improvements, iCLIP enhanced the capacity of CLIP to thoroughly amplify all types of cDNAs (rather than just those cDNAs that read through the crosslink sites), and enabled the sites of RNA-protein interactions to be quantified precisely and at high resolution by analysing the starting position of truncated cDNAs after their genomic mapping, and quantifying their UMIs with software called "iCount". Almost all variants of CLIP that were more recently developed (such as eCLIP, irCLIP and others) have adopted these innovations of iCLIP. Alternative approaches to identify protein-RNA crosslink sites include the mutational analysis of read-through cDNAs, such as nucleotide transitions in PAR-CLIP, or other types of errors that can be introduced by reverse transcriptase when it reads through the crosslink site in standard HITS-CLIP method with the Crosslink induced mutation site (CIMS) analysis. An improved variant of iCLIP (iiCLIP) recently became developed to improve the efficiency and convenience of cDNA library preparation, for example by enzymatically removing adaptor after ligation to minimise artefacts caused by adaptor carry-over, introducing the non-radioactive visualisation of the protein-RNA complex (as done originally by irCLIP), increasing efficiency of ligation, proteinase and reverse transcription reactions, and enabling bead-based purification of cDNAs.

Analysis of CLIP sequencing data benefits from use of customised computational software, much of which is available as part of the Nextflow pipeline for CLIP analysis, and specialised software is available for rapid demultiplexing of complex multiplexed libraries, comparative visualisation of crosslinking profiles across RNAs, identification of the peaks of clustered protein-RNA crosslink sites, and identification of sequence motifs enriched around prominent crosslinks. Moreover, iMaps provides a free CLIP analysis web platform and well-curated community database to facilitate studies of RNA regulatory networks across organisms, with a backend based on the Nextflow pipeline. It is applicable to the many variant protocols of CLIP (such as iCLIP, eCLIP, etc), and can be used to analyse unpublished data in a secure manner, or to obtain public CLIP data in a well-annotated format, along with various forms of quality control, visualisation and comparison. Questions on the experimental and computational challenges are collated on the Q&A CLIP Forum.