Isotope-free mapping of protein-RNA interactions at single-nucleotide resolution by iCLIP3

Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) enables transcriptome-wide mapping of RNA-binding protein (RBP)-RNA interactions. Here, we present iCLIP version 3 (iCLIP3), a streamlined protocol optimized for generating high-quality iCLIP libraries from low-input material. We describe steps for near-infrared visualization of RBP-RNA complexes, silica column-based RNA isolation, and unique dual indexing using TruSeq adapters for cost-effective multiplexing and sequencing. We detail a complete bioinformatics workflow to identify crosslinking events and define RBP binding sites from raw sequencing data.