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

ABSTRACT

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 transcriptome-scale 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.