GSM2758648: ChIP-Seq_EZH2; Homo sapiens; ChIP-Seq
HEK293T cells were cultured to 80-90% confluence, reaching approximately 30 million cells per 15 cm plate. The plate was washed once with PBS before adding 10 mL of fixative solution (50 mM HEPES pH 7.5 at 25 °C, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1 M NaHCO3, 1% Formaldehyde Methanol free – Thermo Fisher Scientific) and incubating for 6 minutes at 25 °C while shaking gently. Directly afterwards, glycine was added to 125 mM and the plate was incubated for an additional 5 minutes. Cells were scraped off, pelleted at 4 °C, resuspended in 5 mL PBS and 15 mL water and 5 mL 5x nuclear isolation buffer (1.28 M sucrose, 40 mM Tris pH 8 at 4 °C, 20 mM MgCl2, 5 mM EDTA, 2.5 mM EGTA, 4% Triton X100, 1 µg/mL each for Leupeptin, Pepstatin A, Aprotinin) were added. The resulting suspension was rotated at 4 °C for 10 minutes. Nuclei were pelleted and resuspended in 1 mL of sonication buffer (10 mM Tris pH 8 at 4 °C, 1 mM EDTA, 0.1 % SDS, Roche cOmplete protease inhibitors) kept on ice for 5 minutes, transferred to a milliTube (Covaris) and sonicated in a S220 (Covaris) for 12 minutes at 5 % duty cycle, 200 cycles per burst and 140 W max incidence power. Sample was diluted with 250 µL 5x ChIP dilution buffer (10 mM Tris pH 8 at 4 °C, 1 mM EDTA, 0.1 % SDS, 750 mM NaCl, 5 % Triton X-100, 0.5 % Na-Deoxycholate, Roche cOmplete protease inhibitors). The mix was kept on ice for 5 minutes and cleared at maximum speed in a tabletop centrifuge. The supernatant was transferred, aliquoted and flash frozen in siliconized epis using liquid nitrogen.  For each IP one aliquot was thawed on ice and pre-cleared with 10 µL of protein A or G dynabeads (Thermo Fisher Scientific) washed three times with wash buffer (10 mM Tris pH 8, 1 mM EDTA, 150 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 0.1 % Na-Deoxycholate). After 15 minutes at 4 °C the beads were removed and 2 µg of the respective antibody added. The IP was performed overnight at 4 °C while rotating. Next 10 µL of washed protein A or G dynabeads were added and incubated at 4 °C for 4 hours.   After the IP the beads were washed 5 times with 400 µL of wash buffer, once with 400 µL high salt wash buffer (10 mM Tris pH 8, 1 mM EDTA, 500 mM NaCl, 1 % Triton X-100, 0.1 % SDS, 0.1 % Na-Deoxycholate) and once with 400 µL TE, each for 8 minutes at 4 °C. Next the Protein-DNA was eluted from the beads by resuspending the them in 100 µL elution buffer (1 % SDS, 100 mM NaHCO3) and incubated at 65 °C and 1000 rpm in a thermomixer. The elution was repeated twice and to the resulting 300 µL eluate 12 µL 5 M NaCl were added and de-crosslinked at 65 °C overnight.   To remove RNA from the de-crosslinked DNA, 8 µL of RNase A (Roche) was added and the sample was incubated for 1.5 hours at 37 °C. Afterwards, 3 µL of 20 mg / mL Proteinase K (Roche) was added and incubated at 55 °C for 1 hour. The DNA was purified using QIAquick PCR purification kit (Qiagen). Kapa Hyper Prep
GEO Accession
GSM2758648