Human on targetplus druggable genome sirna library

The Dharmacon® human ON-TARGETplus druggable genome siRNA library (7,518 genes total with pools of four unique siRNAs targeting each gene) was aliquoted in triplicate into black, clear-bottom, 384-well plates (Greiner, 781091) for a final diluted siRNA concentration of 25 nM. Control siRNAs targeting JAM-A (receptor for reovirus), reovirus μ2, reovirus μNS, and a non-targeting control were seeded into empty wells. As a control for transfection efficiency, wells also were seeded with the AllStars Cell Death control siRNA (Qiagen). Lipofectamine® RNAiMax transfection reagent was diluted in Opti-MEM reduced-serum medium, and aliquoted into each well (0.07 μL RNAiMax per well). Following a 15-min incubation at room temperature (RT), the siRNA/lipid solution was combined with 5 × 10² HBMECs per well in complete RPMI medium. Cells were incubated at 37°C for 48 h to allow efficient target knockdown.