Metafer software

HeLa WT, HeLa ATRX KO, U2OS^ATRX, and 82-6 cells were transfected with siCtrl or siRECQ5 and incubated with 50 µM BrdU for 48 h. Cells were irradiated with 2 Gy and then, 3 h prior to fixation, were treated with 20 mg/mL caffeine and 10 mg/mL colcemid to enrich for mitotic cells. HeLa cells were collected after 8 h post IR and U2OS^ATRX cells were collected after 16 h due to slower exit from G2 into M phase (SI Appendix, Fig. S2C). The 82-6 cells were treated with 100 nM of the Chk1 inhibitor UCN-01 for 3 h instead of caffeine and collected at 12 h post IR to obtain sufficient mitotic cells. For preparation of chromosome spreads, cells were collected and resuspended in prewarmed 75 mM KCl for 30 min at 37 °C. Cells were then fixed with ice-cold methanol:acetic acid (3:1) three times and chromosomes were spread onto coverslips and air dried overnight. To obtain the differential staining between the two sister chromatids, slides were washed, incubated with 5 µg/mL bisbenzimid (Hoechst 33258) for 1 h, and then covered with 200 mM Na₂HPO₄ and 4 mM citric acid buffer (pH 7.1 to 7.2) and irradiated with ultraviolet light C at 9 J/cm². Slides were then washed in 2× saline-sodium citrate buffer at 50 °C for 30 min and stained with Giemsa. Chromosome spreads were captured and analyzed using a Zeiss microscope with Metafer software (MetaSystems).