RNAPII Ser-2P CUT&Tag assay following osmotic stress and MLN4924 treatment
Osmotic stress triggers Ser2-phosphorylated RNA polymerase II (RNAPII Ser-2P) ubiquitylation and degradation, mediated by Cullin E3 ligase complexes where activated CUL1-FBXO11 complex showed prominent role. Pan-Cullin E3 ligase inhibitor MLN4924 treatment totally reversed osmotic stress induced RNAPII degradation, consequently, osmotic stressed 293T cells (120mM Kcl for 2 hours), who are pretreated with 0.01mM MLN4924 for 2 hours, were utilized to perform RNAPII Ser-2P specific CUT&amp;Tag assay. NT (PBS, DMSO), Kcl (120mM Kcl, DMSO) and MLN4924 (PBS, 0.01mM MLN4924) groups were all conducted as controls, and each group possesses 3 biological replicates. Dissecting genome wide RNAPII Ser-2P binding offers the landscape of osmotic stress induced transcription changes and RNAPII pool size alteration effects on global transcription.