GSM8605994: H1, WT, R1, EZH2, ChIP; Homo sapiens; ChIP-Seq
1 x 106 million cells were double crosslinked with 2mM di (N-succinimidyl) glutarate DSG (45min) and 1% formaldehyde (15 min) at RT. The cells were lysed with a mild lysis buffer (0.1% SDS, 1% Triton, 0.15M NaCl, 1mM EDTA, 20mM Tris) and sonicated using a Qsonica Q700 prove sonicator (7 min ON, 1 min ON/OFF, 25% amp). Following sonication, the non-sonicated chromatin was removed by centrifugation. 300ul of chromatin extract was used per IP, and the  cell lysate was incubated with the 1-5 ug primary antibody overnight at 4°C. After overnight incubation, 10µl of protein A/G magnetic beads was added to the chromatin extracts for 4 hours at 4°C. After four hours, the IPs were washed four times (Wash buffer 1: 0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM HEPES. Wash buffer 2: 0.1% SDS, 0.1% NaDOC, 1% Triton X- 100, 0.5 M NaCl, 1 mM EDTA, 20 mM HEPES. Wash buffer 3: 0.25 M LiCl, 0.5% NaDOC, 0.5% NP-40, 1 mM EDTA, 20 mM HEPES. Wash buffer 4: 1 mM EDTA, 20 mM HEPES), and reverse crosslinked with elution buffer (1% SDS, 0.1 M NaHCO3) overnight at 65°C. The next day, DNA was purified using the Qiaquick PCR purification kit. The DNA was quantified using a Qubit Flex and the libraries were prepared using the NEBNext DNA Library kit. The quality of the libraries was analyzed using the Agilent 4150 Tapestation. Samples were sequenced using a P2 flow cell and NextSeq 1000 instrument.