Quantification of DNA End Resection

DNA end resection was investigated by measuring chromatin-bound RPA as previously described38 (link)45 (link). Briefly, siRNA (control and WRN) transfected U2OS cells were treated with 1 μM camptothecin (CPT) for 1 h. Harvested cells were treated with 1 ml of 0.2% Triton X-100 in PBS on ice for 7 min and washed with 1 ml Perm/Wash buffer (BD Biosciences) and fixed with 300 μl of Cytofix/Cytoperm buffer (BD Biosciences) for 15 min. Following fixation, cells were washed with Perm/Wash buffer for 30 min and treated with 1 μg ml⁻¹ RPA antibody (NA-18, EMD Millipore) in BD Perm/Wash buffer for 1 h at 37 °C and later with 1:500 goat anti-mouse Alexa Fluor 488 (Life Technologies) at 37 °C for 30 min. Immunostained cells were washed with Perm/Wash buffer and suspended in 0.3 ml PBS containing 10 μg/ml propidium iodide (Sigma), 250 μg/ml RNase A (Thermo Fisher) and 0.02% sodium azide (Sigma) for 15 min at 37 °C. Chromatin-bound RPA-positive cells were detected with Accuri C6 Flow Cytometer (BD Biosciences) and analysed with FlowJo v10 (FlowJo). The data represents average of three biological experiments with SEM. P values were calculated with Student t-Test.