Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins

Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP key steps in the PAR-CLIP procedure must be carefully optimized. Here we present an advanced step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. We highlight the use of PAR-CLIP in combination with our computational analysis pipeline by reviewing three PAR-CLIP datasets of RBPs targeting different classes of cellular RNAs.