Photoactivatable-Ribonucleotide-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) of the RNA-binding protein (RBP) RBM4 in KBM-7 derived near-haploid human cell line, HAP1.

We hypothesized that RBM4 regulates HERVs by directly binding to their transcripts. To test this possibility, we performed photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP). We performed four independent PAR-CLIP replicates of our own using HAP1 cells stably expressing a FLAG-tagged RBM4 (FLAG-RBM4) transgene under control of a doxycycline-inducible promoter. Following metabolic labeling with 4-thiouridine (4SU) and crosslinking with ultraviolet light (UV) of 312 nm wavelength, we isolated RNA covalently linked to FLAG-RBM4. The RNA recovered from four biological replicates was converted into cDNA libraries and deep sequenced. Overall design: Four replicate pairs of RBM4 PAR-CLIP libraries were sequenced. For each pair, one library was generated from a FLAG-RBM4 HAP1 sample treated with doxycycline (RBM4 induced) while the other one, as a Control, was not (no RBM4 induction). Dox-treated RBM4 ("RBM4_#_", 4 replicates). noDox (noDox_RBM4_#_", 4 replicates).