RNAPolII CUT&Tag profiles of MDA-MB-231 cell line with repression of CDK genes and PRMT5
A clonal doxycycline inducible dCas9-KRAB MDA-MB-231 cell line to control repression of CDK genes and PRMT5. On day 1 of the experiment cells were infected with lentiviruses containing the appropriate targeting/NTC sgRNAs driven by the human U6 promoter at an MOI of ~3 for each virus to ensure all cells were transduced. Cells were transduced in DMEM + 10% FBS with the addition of 8 µg/mL polybrene. 16 hours after the time of transduction, media was changed to DMEM +10% FBS. 24 hours after this, the cell culture media was switched to DMEM + 10%FBS containing 2µg/mL puromycin to ensure no uninfected cells remain. 48 hours after this, cell culture media was changed to DMEM + 10%FBS containing 2 µg/mL puromycin and 1 µg/mL doxycycline to induce dCas9-KRAB expression. 48 hours after this, cells were processed for CUT&Tag library prep following the manufacturer's recommendations. Overall design: CUT&Tag was used to examine the location of RNA Polymerase II around transcript start sites in MDA-MB-231 cells upon repression of CDK7, CDK9, CDK12, with and without simultaneous repression of PRMT5.