RNase A treatment increases the recruitment of p65 to DNA

We investigated the ability of the NFkB protein p65 to bind DNA after RNA digestion. In this ChIP-seq experiment, we investigated the genome-wide binding profiles of p65 in Jurkat cells. We demonstrated that p65 binding to promoters and promoter proximal regions is enhanced by digestion of RNA with RNase A. p65 ChIP (chromatin immunoprecipitation) libraries were prepared from Jurkat cells. Three experimental conditions were used for cells: DMSO, 200ng/ml PMA and 300ng/ml ionomycin (3h) stimulated and stimulated and 2mg/ml PureLink Rnase A treated. Cells were cross-linked with 1% parafolmaldehyde for 10 minutes and glycine was added to quench the reaction. The cell lysates were prepared as described (Varshney et al., 2018). The chromatin was sonicated in Bioruptor (Diagenode) at high power for 40 min (30 s on/off). 40 micrograms of the sonicated chromatin were used for each IP. 5% of each extract used in the IPs were taken as inputs.For p65, 7.5ug of anti-p65 antibody or rabbit polyclonal IgG antibody were added to samples and incubated overnight at 4oC, before incubation with 30ul Protein G Dynabeads (Life Technologies) for an additional 2h. Beads were eluted as desribed by Varshney et al before Proteinase K treatment and reverse crosslinking at 42oC overnight. Eluted DNA was purified by QIAquick PCR purification kit (QIAGEN).