U2OS osteosarcoma cells were cultured in DMEM supplemented with 10% fetal bovine serum (Sigma) at 37°C, 5% CO2. Cells were seeded in 35 mm glass-bottom dishes (MatTek) at 100,000 cells per dish and imaged 48 h after seeding. U2OS cells stably expressing PAGFP-H2A (Bonin et al., 2018 blue right-pointing triangle) were used to track chromatin microdomains. For single-nucleosome imaging, we generated U2OS cells stably expressing H2B fused to the HaloTag by transfection of the pBREBAC-H2BHalo plasmid (Addgene plasmid #91564) using Lipofectamine 3000 (ThermoFisher) followed by clonal selection with geneticin. Before live-cell imaging, H2B-HaloTag U2OS cells were incubated with 10 pM fluorescent JF 459 HaloTag ligand (Grimm et al., 2015 blue right-pointing triangle) for 1 h, washed three times with phosphate-buffered saline, and incubated in DMEM without phenol red for at least 30 min. This concentration of dye proved to be optimal for imaging and tracking. For fixed imaging, cells were imaged after fixation with Formalin (Sigma #HT5011; 20 min).

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