DNA Fiber Assay for Replication Dynamics

DNA fiber assay was performed as described in Bellelli et al. (2018) (link). Briefly, Rtel1^F/FPole4^+/+and Pole4^−/− MEFs infected with CRE recombinase or GFP expressing adenovirus, were pulse labeled with 20 μM CldU for 20 min and subsequently with 200 μM IdU for 20 min. Cells were trypsinized, washed in PBS and resuspended at a concentration of 5x 10⁵ in PBS. 2.5 μL of cell suspension were spotted on clean glass slides and lysed with 7.5 μL of 0.5% SDS in 200 mM Tris-HCL, pH 7.4, 50 mM EDTA for 10 min at R.T. Slides were then tilted allowing a stream of DNA to run slowly down the slide, air-dried and then fixed in methanol/acetic acid (3:1) for 15 min at R.T. After denaturation in HCl 2,5 M (30 min R.T.) slides were blocked in 1% BSA/PBS and incubated with rat anti-BrdU monoclonal antibody (1:1000 overnight; AbD Serotec) and mouse anti-BrdU monoclonal antibody (1:500 1h R.T.; Becton Dickinson). After washes in PBS, slides were incubated with Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500 R.T.; Invitrogen) for 45 min and mounted in PBS/Glycerol 1:1. Fibers were then examined using Axio Imager.M2 (ZEISS) with 60x oil immersion objective and the Volocity 6.3 software.