The NFkB protein RELA/p65 binds predominantly transcript introns and exons

We investigated the ability of the NFkB protein p65 to bind RNA and what its target transcrips were. In this CLIP-seq experiment we demonstrated that p65 is capable of binding RNA and it has a preference to introns and exons, suggesting it binds unspliced RNA. Overall design: p65 and IgG seCLIP (single-end crosslinking and immunoprecipitation) libraries were prepared from Jurkat cells. Cells were UV cross-linked at 254 nM with an energy setting of 400 mJ/cm2. IP, RNA isolation and library preparation was performed as described by Van Nostrand et al, 2018. Lysates were treated with RNase I diluted 1:150. IPs were performed with 7.5µg anti-p65 Santa Cruz antibody sc-8008 or 7.5µg mouse IgG. The chromatin was sonicated in Bioruptor (Diagenode) at low power for 5 min (30 s on/off). Antibodies were tethered to protein G magnetic beads for 1h at room temperature and afterwards, the lysates were incubated with the beads. The next day 2% of lysate+bead was set aside as input. Library prep proceeded as described in Van Nostrand et al, 2018. RNA was purified using Zymo RNA Clean and Concentrator-5. DNA libraries were extracted from agarose gels in the end using a gel extraction kit (QIAGEN). Libraries were quantified using D1000 Agligent TapeStation.