High-throughput siRNA screening

The siRNA library screening was performed at the University of Washington Quellos High-Throughput Screening Core using the previously published DDR322 siRNA library (Kehrli et al., 2016). The DDR 322 siRNA library consists of commercially obtained three independent siRNAs that target each of 322 genes involved in double strand DNA break repair (cat. no. 1027416, Qiagen, Germantown, MD) (Kehrli et al., 2016). For siRNA screening, 200 cells were plated per well in 384-well plates, and 24 h later, cells were transfected with the siRNA library using DharmaFECT1 (cat. no. T-2001, Dharmacon, Lafayette, CO) (Kehrli et al., 2016). Transfections of three independent siRNAs per gene per well were done in triplicate with a mock control and universal siRNA control in each plate. Transfection efficacy was monitored by the toxicity of Kif11 siRNA controls placed in each plate. 24 h later, hydroxyurea was added at final concentrations of 1 or 2 mM. Two days later, relative cell numbers were determined by the CellTiter-Glo Assay (cat. no. G7570, Promega, Madison, WI) according to the manufacturer's instructions. The signal of siRNA-transfected wells were normalized to mock transfected wells to obtain the relative cell number. Student's t-test was performed to test mean by well and treatment/vehicle mean by well and treatment. Pairwise p-values were calculated using test triplicates versus vehicle triplicates.