Zymolyase 100t

Cells were fixed in 3% paraformaldehyde EM grade (Electron Microscopy Sciences) for 15 min at RT. Cells were washed three times in PEM (100 mM Pipes, 1 mM EGTA, 1 mM MgSO4 pH = 6.9) and once in PEMS (PEM plus 1.2 M Sorbitol). Cells were treated with 0.5 mg/mL Zymolyase-100T (Seikagaku) for 1 h at 37°C. Cells were permeabilized in PEMS plus 0.5% Triton X-100 and incubated in ice for 10 min. Cells were then washed in PEM and incubated in PEMBAL (PEM + 1% BSA, 100 mM Lysine hydrochloride, 0.1% NaN3, pH = 6.9) containing 5 mM MgSO4 for 30 min. BrdU epitopes were exposed by incubating cells for 2 h at 37°C in PEMBAL containing 5 mM MgSO4, nucleases from the 5-Bromo-2′-deoxy-uridine labeling and detection kit I (Roche), and mouse anti-BrdU antibody (BD Biosciences, 347580). Cells were then washed three times with PEMBAL and incubated overnight with mouse anti-BrdU antibody (BD Biosciences, 347580). Cells were then washed three times in PEMBAL, incubated with Alexa Fluor 488 goat anti-mouse antibody (Invitrogen, A-11029) for 3 h, washed in PEMBAL, in PBS, and stained with DAPI. Alternatively, for heat denaturation, after permeabilization, cells were recovered in PEMS and incubated for 10 min at 95°C, followed by incubation in an ice/H2O bath for 5 min.

Chromatin association assays were performed in triplicate as previously described [87 (link)]. Overnight cultures were diluted to 0.2 OD₆₀₀ in 50 mL of SC-TRP and grown to 0.5 OD₆₀₀. A total of 25 OD units were harvested, incubated in Pre-spheroplast Buffer (100 mM PIPES/KOH [pH 9.4], 10 mM DTT, 0.1% sodium azide) for 10 minutes at room temperature, and spheroplasted with 20 mg/mL Zymolyase-100T (Seikagaku Corporation) in Spheroplast Buffer (50 mM KPO₄ [pH 7.5], 0.6 M sorbitol, 10 mM DTT) for 30 minutes at 37°C. The resulting spheroplasts were washed with Wash Buffer (50 mM HEPES/KOH [pH 7.5], 100 mM KCl, 2.5 mM MgCl₂, 0.4 M sorbitol), resuspended in EB (50 mM HEPES/KOH [pH 7.5], 100 mM KCl, 2.5 mM MgCl₂, 1 mM DTT, 1 mM PMSF, 1 μg/mL leupeptin, 2 μg/mL aprotinin, 1 mM PMSF) and lysed on ice with 0.1% Triton X-100. A portion of the resulting whole cell extracts (WCE) were saved, while the remaining lysate was centrifuged through EBSX (EB + 0.25% Triton X-100 and 30% sucrose) to separate the chromatin pellet and supernatant fractions. The WCE, pellet, and supernatant fractions were analysed using SDS-PAGE followed by immunoblotting with anti-FLAG M2 (Sigma), anti-H4 (Abcam), and anti-PGK1 (Sigma) antibodies. Bands were visualized using Odyssey Infrared Imaging System (LI-COR).

Cells were mounted on a glass-bottom dish (MatTek) coated with concanavalin A and covered with fixative [2% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2)]. After 1 min, cells were further fixed with fresh fixative for 2 hr at 4°C. After washing with buffer, low melting agarose was applied onto the cells to prevent loss of cells during subsequent procedures. Zymolyase solution (0.4 mg⁄ml zymolyase 100T, Seikagaku Co., Tokyo, Japan) was applied on top of the agarose for 60 min at 37°C, postfixed with 2% OsO₄ for 2 hr at room temperature, stained with 1% uranyl acetate for 1 hr, dehydrated with acetone in an ascending series from 50% to 100%, and embedded in epoxy resin. Serial sections of 80 nm thicknesses were obtained, poststained with uranyl acetate and lead citrate, and analysed using a Zeiss EM900 Transmission Electron Microscope at Central Unit Electron Microscopy in the German Cancer Research Center (DKFZ) (ZEISS, Oberkochen, Germany) or a Hitachi H-7500 Transmission Electron Microscope at Research Centre for Ultra-High Voltage Electron Microscopy at Osaka University (Hitachi, Tokyo, Japan).