DNA fiber assay

DNA fiber assays were performed as previously described⁵. Briefly, cells were pulse labeled with 50 µM IdU and 50 µM CldU, untreated or treated with 4 mM HU, as indicated. 2000–4000 cells were lysed in lysis buffer (0.5% SDS, 200 mM Tris-HCl pH 7.4, 50 mM EDTA). DNA fibers were spread on microscope slides and fixed in methanol/acetic acid (3:1 by volume). DNA was denatured in 2.5 M HCl for 30 min, followed by 1 h blocking buffer (10% goat serum and 0.1% Triton-X in PBS). Slides were incubated with primary antibodies, anti-CldU (1:75; MA1-82088, Thermo Fisher Scientific) and anti-IdU (1:75; 347580, BD Biosciences) followed by secondary antibodies, anti-rat Alexa Fluor® 488 and anti-mouse Alexa Fluor® 594 (1:250, Thermo Fisher Scientific), for 1 h each in blocking buffer at room temperature. Slides were mounted in Prolong with DAPI ({"type":"entrez-protein","attrs":{"text":"P36935","term_id":"549826","term_text":"P36935"}}P36935, Thermo Fisher Scientific) before image acquisition under Axio2 microscope (Zeiss). Images were analyzed with FIJI (ImageJ) software.