All cells were prepared by previously published chromEM staining protocol (8) and described in detail in section S2. Two kinds of sections were made using an ultramicrotome (UC7, Leica). For the tomography, 100- to 200-nm-thick resin sections were cut and deposited onto a copper slot grid with carbon/formvar film (EMS). For investigating the chromatin structure difference with and without DXM treatment, 50-nm-thick resin sections were made and deposited onto copper 200-mesh grid with carbon/formvar film (EMS). The grids were plasma-cleaned by a plasma cleaner (easiGlow, TED PELLA) before use. No poststaining was performed, but 10-nm colloidal gold particles were added to the tomography samples on both sides as fiducial markers.

A 200-kV STEM (HD2300, HITACHI) was used for tomography data collection. HAADF imaging contrast was used in the tilt series. To reduce the missing wedge, tilting series from −60° to 60° on two perpendicular axes were recorded manually, with 2° step size. The pixel dwell time was kept small (~5 μs) to prevent severe beam damage during imaging. For the thin sections, a TEM (HT7700, HITACHI) was operated at 80 kV in the bright field to capture high-contrast chromatin data.

For tomography, a combination of methods was used to achieve high-quality reconstruction (section S2). A nominal voxel size of 2 to 2.9 nm was used in the tomography to resolve individual nucleosomes. The DNA density was used to generate color-coded nucleosome configurations, with green color dictating the lowest density, and red dictates the highest density. The chromatin binary masks were used to generate the surface of supranuclesomal structures. The videos of example tomography and volume rendering can be found in movies S4 and S5.

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