Reverse Transfection for RNAi Depletion

RNAi depletion by reverse-transfection was conducted in MCF10A cells, hTERT RPE-1 cells, and HeLa cells as previously reported (54–56 (link)). Briefly, cells were reverse-transfected into an arrayed 384-well plate library containing small interfering RNA (siRNA) or miRIDIAN microRNA mimic constructs (Horizon Discovery, Supplementary Tables S1-S7) using Opti-MEM (Gibco 31985070) and Lipofectamine RNAiMAX transfection reagent (Invitrogen 13778150). Assay-ready plates containing 10 μl of 100 nM microRNA mimics resuspended in 1X siRNA buffer (Horizon Discovery B-002000-UB-100) were prepared from master library 384-well plates (Horizon Discovery, 0.1 nmol scale) and stored at −80°C. Plates were prepared with control siRNAs (siNT, siNOL11, siKIF11 or siPOLR1A) for reverse-transfection at a final 20 nM siRNA/microRNA mimic concentration as described (56 (link)), at a seeding density of 3000 MCF10A or HeLa cells/well or 4000 hTERT RPE-1 cells/well.