RPB1 immunofluorescence in HCT116 cells (ARMC5 knockdown) treated with transcriptional inhibitors

siRNA transfections. In 96-well plates, 25 µL of siRNA at 30 nM in Opti-MEM (Thermo Fisher 31985-062) was added per well, followed by 25 µL of transfection reagent (Thermo Fisher Lipofectamine RNAiMAX 13778100) diluted 1/125 in OptiMEM. In 384-well plates, 10 µL of siRNA at 30 nM in Opti-MEM was added per well, followed by 10 µL of diluted transfection reagent.

Cells were fixed in 4% paraformaldehyde (EMS Emgrid 15710) for 15 minutes, then permeabilised in 0.25% Triton X100 (Sigma Aldrich 93443) for 10 minutes. Cells were incubated in 50% blocking buffer (Millenium Biosciences Li-Cor Intercept in PBS, LCR-927-70001) in PBS for 30 minutes, before being stained with primary antibodies in 50% blocking buffer in PBS for 90 minutes. Cells were then incubated for 30 minutes with secondary antibodies plus DAPI at 200 ng/mL in 50% blocking buffer in PBS.

Imaging was performed on a Perkin Elmer Operetta CLS, with 40x/NA1.1 water immersion objective and LED light source.