Protocol for detecting RBM33-binding sites in HEK293T cells using PAR-CLIP-seq

RNA -binding proteins (RBPs) regulate gene expression both co -transcriptionally and post -transcriptionally. Here, we provide a protocol for photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation followed by next -generation sequencing (PAR-CLIP-seq). PAR-CLIP-seq is a transcriptomescale technique for identifying in vivo binding sites of RBPs at the single -nucleotide level. We detail procedures for the establishment of FLAG-RBM33 stable cell line, the sequencing library preparation, and the data analysis.