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 optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data analysis pipeline to three distinct RBPs targeting different annotation categories of cellular RNAs.