siRNA screening procedure
Screens were performed in 384-well plates using reverse transfection. Per well, 1 pmol siRNA dissolved in 5 µl H2O was supplemented with transfection reagent (0.0625 µl Oligofectamin (Invitrogen) in 10 µl Opti-MEM (Life technologies) for the primary screen; 0.125 µl INTERFERin (Polyplus transfection) in 10 µl Opti-MEM for validation screens). Plates were incubated for 30 min at RT. Then, 60 µl of HeLa RPS2-YFP cells (8000 cells/ml in DMEM+/+) were added. Plates were incubated for 58 h. Expression of the RPS2-YFP reporter was induced for 14 h by addition of 10 µl DMEM+/+ supplemented with tetracycline (final concentration 125 ng/ml). Cells were fixed by addition of 20 µl 16% PFA in PBS. After 10 min, cells were washed once with PBS, incubated with Hoechst in PBS for 10 min, washed again and kept in H2O/NaN3. From each well, 9 pictures were taken by automated microscopy using an MD microscope.