GSM9261502: UNC_H2AK119ub_Rep1; Homo sapiens; OTHER
Cells were harvested by incubation with TrypLE for 5-7 minutes at 37C. TrypLE was quenched by wash buffer (0.1 % BSA in DMEM/F12) and cells were collected by centrifugation at 300 x g for 3 min. Cells were washed in 1X PBS without Calcium or Magnesium (PBS -/-) and collected by centrifugation at 300 x g for 3 minutes. Cells were resuspended in PBS -/- before addition of 2X nuclear extraction buffer (25 mM HEPES pH 7.5 (Gibco, Cat. 15630), 12.5 mM Potassium Chloride, 0.25 mM Spermidine, 0.13 % Triton X-100 (Sigma, Cat. T9284), 25% Glycerol  and 1 x Protease inhibitor cocktail (Roche) in water) to 1X and incubation on ice for 10 minutes with intermittent vortexing. Nuclei were collected by centrifugation at 1300 x g for 4 min at 4 °C. Nuclei were resuspended in cold PBS -/- and fresh 16% PFA was added to a final concentration of 0.1% followed by rocking gently at room temperature for 2 min. Fixation was stopped by addition of 1.25 M glycine. Nuclei were collected by centrifugation at 1300 x g for 4 min at 4°C and resuspended in Wash buffer. Human cells (n = 20,000) were mixed with Drosophila S2 cells (n = 8,000) per reaction prior to addition of concanavalin-beads. Concanavalin A beads (10 µL per 100,000 cells; Stratech, Cat. BP531-BAN-3ml) were first activated in Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 10 mM CaCl₂, 10 mM MnCl₂) and incubated with cells at room temperature for 10 min with agitation. Bead-cell complexes were resuspended in Antibody Buffer (2 mM EDTA pH 8, 0.1 % BSA in Dig-Wash Buffer) containing primary antibodies (1:100) and incubated overnight at 4 °C on a tube rotator. The following antibodies were used at a 1:50 dilution H3K27me3 (CST, Cat. 9733) and H2AK119Ub (Cell Signalling, Cat. D27C4). After a wash in Dig-Wash Buffer (0.05 % digitonin in Wash Buffer), complexes were incubated with a secondary antibody (guinea pig α-rabbit IgG, 1:100; Rockland, Cat. ABIN101961) for 30 min at room temperature on a tube rotator. After three washes in Dig-Wash Buffer, bead-cell complexes were incubated with pAG-Tn5 (1:250; EpiCypher, Cat. 15-1117) in Dig300-Wash Buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM spermidine, protease inhibitors, 0.01 % digitonin) for 1 h at room temperature. After three washes in Dig300-Wash Buffer, tagmentation was activated in Tagmentation Buffer (10 mM MgCl₂ in Dig300-Wash Buffer) for 1 h at 37 °C. The tagmentation reaction was stopped by the addition of 10 µL 0.5 M EDTA, 3 µL 10 % SDS, and 2.5 µL 20 mg/mL proteinase K, and incubated for 1 h at 55 °C. DNA was purified with 1 X AMPure XP beads (Beckman Coulter) to enrich for ≥ 100 bp fragments. Libraries were amplified using KAPA HiFi HotStart polymerase (Roche Sequencing Store, Cat. KK2601) with 14 and 18 PCR cycles for H3K27me3 and H2AK119ub, respectively.