CLIPSeq

CLIP-seq, short for crosslinking and immunoprecipitation followed by sequencing, is a set of experimental approaches used to map interactions between RNA-binding proteins (RBPs) and their RNA targets in living cells. The technique combines in vivo crosslinking of RNA and protein with immunoprecipitation of the RBP and high-throughput sequencing of the co-purified RNA fragments, enabling genome-wide identification of binding sites and target transcripts.

The typical workflow involves crosslinking RNA-protein complexes with ultraviolet light to create covalent bonds, lysing cells, and partial digestion of RNA. The RBP of interest is then immunoprecipitated, along with its crosslinked RNA, and the bound RNA fragments are purified, ligated to adapters, reverse-transcribed, and sequenced. The resulting reads are aligned to a reference genome or transcriptome to locate protein-binding sites. Some variants produce additional information, such as single-nucleotide resolution of crosslink sites or characteristic sequence changes that aid site localization.

Major variants include HITS-CLIP (high-throughput sequencing CLIP), PAR-CLIP (photoactivatable-ribonucleoside-enhanced CLIP), and iCLIP (individual-nucleotide resolution CLIP). PAR-CLIP incorporates photoactivatable nucleosides to increase crosslinking efficiency and leaves detectable mutations in reads, while iCLIP uses reverse transcription truncations to infer the exact crosslink position. Enhanced versions like eCLIP further improve efficiency and reproducibility.

Applications of CLIP-seq span mapping global RNA-protein interactions, identifying binding motifs, studying post-transcriptional regulation, and characterizing targets of Argonaute and other RBPs. Limitations include crosslinking efficiency biases, antibody quality, digestion variability, and the need for rigorous data analysis to distinguish true binding from background.