Quantitative image‐based cytometry (QIBC)

For the experiments in Figs 2C and ​and4,4, mouse ES cells were seeded at 10,000 cells per well in clear‐bottomed black 96‐well plates (Greiner, 655090). Cells were grown for 24 h before addition of 250 nM AGB1 for 0, 4 or 8 h, followed by treatment with 20 μM EdU for 20 min. To remove proteins not bound to chromatin, cells were pre‐extracted for 5 min with cold CSK buffer (“Cytoskeleton buffer”: 10 mM PIPES pH7, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂) supplemented with 0.5% Triton X‐100, 1 mM CaCl₂ and protease inhibitors (Merck, cOmplete EDTA‐free, 1187358001; one tablet dissolved in 50 ml buffer), before fixation 20 min in 2% formaldehyde. For EdU detection, the far‐red fluorescent dye Alexa‐647 was covalently linked to EdU using the click‐iT EdU Imaging Kit (Thermo Fisher, {"type":"entrez-nucleotide","attrs":{"text":"B10184","term_id":"2091303","term_text":"B10184"}}B10184). To detect CDC45, cells were incubated in methanol for 15 min at 4°C and then blocked with 5% BSA for 30 min, before incubation with a 1:200 dilution of a rabbit monoclonal anti‐CDC45 antibody (Cell Signalling Technology, 11881) for 60 h at 4°C, followed by 30 min at room temperature with donkey anti‐rabbit IgG coupled to Alexa Fluor 488 (Invitrogen, A‐21206), in the presence of 2.5 μg/ml DAPI (Thermo, 62248). For detection of MCM2, cells were processed as for CDC45 but without the methanol fixation step and with a 1:1,000 dilution of mouse monoclonal anti‐MCM2 (BD Biosciences, 610701) for 14 h at 4°C, followed by 30 min at room temperature with donkey anti‐mouse IgG coupled to Alexa Fluor 488 (Invitrogen, A‐21202) together with 2.5 μg/ml DAPI (Thermo, 62248). GFP‐SLD5 was detected using either of the above conditions used for CDC45 or MCM2. Finally, cells were washed and stored in PBS at 4°C until imaging by scanR High Content Screening Microscopy (Olympus). Images were analysed with the ScanR analysis software and data visualised in Tableau.