Bay et al. 2022 Figure 5

To address whether promoter proximal RNAPII is degraded on chromatin after UV by adding DRB prior to UV.
Conclusion:
DRB prior to UV further enhanced removal of RNAPII from chromatin compared to cells treated with DRB or UV alone. The increase in chromatin levels of pRNAPII S5 was greater than the increase in pRNAPII S2 after treatment with UV + DRB + MG132 compared to UV + DRB, in line with more promoter proximal RNAPII being degraded than elongating RNAPII. DRB caused a relatively greater reduction in pRNAPII S2 levels in G1 and G2 phase compared to S phase after UV
Notes:
Cells were treated with 1uM EdU for 30 min prior to inhibitor treatment + during treatment to allow cell cycle analysis. Cells were chromatin extracted prior to fixation to allow release of unbound factors. Secondary antibody controls were included. Barcoded non-treated cells were included to minimize sample to sample variation during staining. In brief, non-treated HeLa cells were incubated with Alexa Fluor 647 Succinimidyl Ester. Barcoded cells were then distitributed equally among all the samples prior to antibody staining. All samples were normalized to the barcoded control upon analysis. CST run prior to analysis.