DNA Fiber Assay for Chromatin Dynamics

DNA fiber assay was performed as described in(Bellelli et al., 2018 (link)). Briefly, eHAP ALC1^+/+ and ALC1^−/− transfected with control siRNA or siRNA targeting BRCA1 or BRCA2, were pulse labeled with 20 μM CldU for 20 min and subsequently with 200 μM IdU for 20 min. After tripsinization and counting, cells were resuspended at a concentration of 5x 10⁵ in PBS and 2.5 μL of cell suspension were spotted on glass slides and lysed with 7.5 μL of a buffer containing 0.5% SDS, 200 mM Tris-HCL, pH 7.4, and 50 mM EDTA. Slides were then tilted to allow a stream of DNA to move slowly toward the botton of the slide, briefly air-dried and then fixed in methanol/acetic acid (3:1) (15 min at R.T). Slides were subsequently denatured in HCl 2,5 M (30 min R.T.), extensively washed in dH2O and PBS, blocked in 1% BSA/PBS (30 min R.T.) and incubated with rat anti-BrdU monoclonal antibody (1:1000 overnight; AbD Serotec) and subsequently with mouse anti-BrdU monoclonal antibody (1:500 1 h R.T.; Becton Dickinson). After incubation with a mixture of Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500 45 min R.T.; Invitrogen) slides were mounted in PBS/Glycerol 1:1 and finally examined using Axio Imager.M2 (ZEISS) with 63x oil immersion objective and the Volocity 6.3 software.