EdU-labeled fiber assays

Nascent DNA of U2OS cells was labeled with 2 µM EdU for 90 min, and cells were then washed twice before releasing into media without EdU for 26.5 h. In the PARPi-treated conditions, 10 µM olaparib was added either 4 h before collecting samples or throughout the experiment including during EdU labeling. Forty minutes prior to collecting samples, cells were labeled with 50 µM CldU for 20 min, washed twice with prewarmed media, and subsequently labeled for 20 min with 100 µM IdU. Importantly, in the PARPi-treated conditions, 10 µM olaparib was added during CldU and IdU labeling as well as in the media used for washes. Cells were then trypsinized and resuspended at ∼1000 cells/µL in PBS. Three microliters of cell suspension was dropped onto slides and allowed to settle for 3 min. Seven microliters of lysis buffer (200 mM Tris-HCl at pH 7.4, 0.5% SDS, 50 mM EDTA) was then gently added, and cell lysis was allowed to occur for 3 min at room temperature. Slides were then tilted at a 15° angle to let cell lysates slowly flow down the length of slides and stretch DNA fibers. Immunofluorescence was then carried out as described in the S1 nuclease assay with few modifications. EdU was biotinylated prior to blocking by incubating slides in PBS with 2 mM CuSO₄, 10 mM sodium ascorbate, and 1 µM biotin azide (Click chemistry tools 1265) for 1 h at room temperature. Biotinylated EdU was detected with a rabbit anti-biotin antibody (Abcam ab53494). Antirabbit AlexaFluor 488, antimouse AlexaFluor 647, and antirat Cy3-conjugated secondary antibodies were used.