Smartpool on target

MRC5 cells were cultured in MEM supplemented with 15% FCS, 1% l-glutamine and 1% PenStrep (v/v), at 37°C in CO₂ controlled incubators. Cells were transfected with PolDIP2 siRNA (SMARTpool ON-TARGET plus siRNA Thermo Fisher Scientific), or mock siRNA treated, using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. 72 h following siRNA transfection cells were subject to DNA fibre analysis as described previously (8 (link)). All DNA fibre analysis was performed in triplicate. MRC5 PrimPol^−/− cells were generated using the zinc finger nuclease knockout method according to the manufacturer's recommendations (Sigma-Aldrich).

For transfection, isolated BMDMs were differentiated with 20 ng/ml M-CSF for 1 week and transfected afterwards with 150 nM SMARTpool ON-TARGET plus siArnt or ON-TARGET plus NON-TARGETing pool siCtrl (both Thermo Scientific, Karlsruhe, Germany) using Hiperfect transfection reagent (Quiagen, Hilden, Germany). Therefore, BMDMs were incubated in medium without FCS containing siRNA and Hiperfect. After 6 h, medium containing M-CSF (final concentration of 20 ng/ml) was added and after 24 h cells were stimulated with MCA (5 μg/ml) or dimethylsulfoxide (DMSO) as a control for 8 h.