GSM6631355: Ring1bflfl-G2; Mus musculus; OTHER
Chromatin extraction and fixation was carried out from 2x106 cells from G1 and G2-sorted FUCCI-Ring1bfl/fl and FUCCI-Ring1b-/- cells. Cells were resuspended in 1.86 ml of complete medium and fixed with formaldehyde (1.89%) and incubated for 10 minutes on a rotating wheel at room temperature. The fixation reaction was stopped by adding cold glycine (final concentration 150 mM). Fixated cells were centrifuged (5 min 1000 rpm 4°C) and washed 1x with cold PBS. After centrifugation, cells were resuspended in cold lysis buffer (10 mM Tris pH 8, 10 mM NaCl, 0.2% NP-40, 1x protease inhibitors (Roche)) and incubated for 20 min on ice. Subsequently, the pellet was centrifuged (5 min, 1800 rpm, 4°C) and resuspended in 200 μl of lysis buffer. Next steps were carry out as described previously in (Dimitrova et al., 2021) 2-4 µg DNA from each sample were adaptor-ligated and indexed using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and NEBNext Multiplex Oligos for Illumina (New England Biolabs). The libraries were amplified with 7 PCR cycles using Herculase II Fusion Polymerase kit (Agilent). Libraries hybridisation was performed as described previously (Dimitrova et al., 2021)