Acumen explorer ex3 laser scanning microplate cytometer

The primary screen was performed using the human siRNA Smart-pool siGenome library from Dharmacon, whereas the secondary screen was performed using the corresponding deconvoluted siRNA duplexes (where four siRNAs included in the pool were tested independently). Contact-inhibited RPE1 cells were reverse-transfected in triplicate 384-well plates using Lullaby transfection reagent [OZ Biosciences, 800 cells/well, 0.2 μL Lullaby/well, 18.75 nmol/L siRNA, 250 nmol/L reversine (Sigma)] using a Biomek FX liquid handling robot (Beckman). Cells were cultured for 96 hours and fixed with 4% formaldehyde (ThermoFisher, no. 28908), except for the deconvolution experiment, where cells were pulsed for 1 hour with 10 μmol/L EdU before fixation to label cells in S phase. Cells were subsequently permeabilized and blocked in PBS + 3% BSA + 0.2% Triton-X100 for 30 minutes and processed for immunofluorescence (IF) and EdU detection (LifeTechnology) according to the manufacturer’s protocol. Images were acquired and analyzed using an ArrayScan VTi-automated microscope (Cellomics), except for total cell counts, which were acquired on an Acumen Explorer eX3 laser scanning microplate cytometer (TTPLabtech). For each parameter, a median Z-score was derived using plate normalization [(well value – plate median)/plate Median Absolute Deviation].