Agarase

The SMART DNA fiber assay procedure was performed largely as described previously (Clairmont et al., 2020 (link)). In brief, cells were treated with BrdU (sigma) for 24h, and then exposed to X-ray irradiation to induce DSB formation. Cells were collected 6h after irradiation, and embedded in low melting point agarose plugs before lysis with proteinase K overnight at 50°C. The plugs were then washed with TE buffer and digested with agarase (NEB). The sample solution was spread onto silanized coverslips using the FiberComb machine (Genomic Vision). Combed coverslips were blocking with 3% BSA for 30min, and then incubated with anti-BrdU antibody (rat, abcam) overnight at 4°C. After incubation with secondary Alexa 555-labelled goat anti-rat antibodies, the coverslips were washed and mounted with Vectashield mounting medium (Vector laboratories). Images were captured by Zeiss AX10 flurorescence microscope. At least 50 fibers were counted per condition. The fiber lengths were measured using ImageJ and graphed.