RPB1 immunofluorescence in HCT116 cells (ARMC5, INTS8 knockdowns)

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. In 8-well chamber slides, 40 µL of siRNA at 30 nM in OptiMEM was added per well, followed by 40 µL of diluted transfection reagent. After 20-30 minutes of room temperature incubation, HCT116 cells were added onto the transfection reaction and allowed to settle. Experiments were conducted three days after siRNA transfection.

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 Nikon Ti2 microscope equipped with a Yokogawa CSU-W1 spinning disk, with 40x/NA0.95 Plan Apo λ air objective, and dual Hamamatsu ORCA-Fusion C14440-20UP cameras. 20 z-planes at 1 µm intervals were acquired. DAPI DNA stain was acquired with a 405 nm laser and 450/82 nm filter. Alexa488+ conjugated secondary antibodies were acquired with a 488 nm laser and 525/50 nm filter, and mCherry-RPB1 was acquired with a 561 nm laser and 617/73 nm filter.