Proteins begin to interact with nascent RNAs as soon as transcription is initiated. The protein complement decorating an RNA molecule changes dynamically in space and time, orchestrating RNA processing and function in the nucleus and cytoplasm 1 . Ribonucleoprotein (RNP) complexes are key to every step of RNA processing and function, and understanding the roles that RNA-binding proteins (RBPs) play requires methods that identify the set of RNAs that they bind in cells during specific developmental stages, activities or disease states.Numerous methods can characterize the RNA interactions that coordinate RNP assembly. These approaches can be protein-centric, describing the compendium of RNA sites bound by a specific RBP, or RNA-centric, identifying the RNA-bound proteome. The most common protein-centric strategies are based on the immunopurification of an RBP and its associated RNAs, and can be broadly categorized as RNA immunoprecipitation (RIP) or cross-linking and immunoprecipitation (CLIP) approaches. RIP approaches purify the RNAprotein complexes under native conditions 2,3 or using formaldehyde cross-linking 4 . CLIP techniques are more widely used and rely on the irradiation of cells by UV light, which causes proteins in the immediate vicinity of the irradiated bases to irreversibly cross-link to the RNA by a covalent bond 5 (Fig. 1). The covalent cross-links allow stringent purification of the RNA-protein complexes, which is followed by a series of steps to determine the interactions of a specific protein across the transcriptome. CLIP uses a limited RNase treatment of cross-linked RNPs to isolate RNA fragments occupied by the RBP and sequencing of these fragments can identify RBP binding sites, which allows inference of RBP function through determining the location of binding sites relative to, for example, other RBP binding sites or cis-acting elements (Box 1).The development of high-throughput sequencing of RNA isolated by CLIP (HITS-CLIP) has enabled a transcriptome-wide view of RNA binding sites 6 . CLIP techniques have been further developed to identify cross-link sites with nucleotide resolution, either through analysis of mutations in reads (photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP)) 7 or by capturing cDNAs that terminate at the cross-linked peptide during reverse transcription (individual-nucleotide resolution CLIP (iCLIP)) 8 . The development of dedicated bioinformatics workflows has allowed the determination of binding sites and consensus motifs to better understand post-transcriptional regulation 9 .This Primer focuses on experimental and computational aspects of CLIP methods that have been broadly adopted and have generated widely used data sets. We also cover the identification of RBP binding sites by tagging RBPs with enzymes that naturally act on RNA, where the resulting RNA modifications can be identified by high-throughput sequencing 10 , as well as the use of
