GSM5939989: differentiated hiPSCs_dMet_H3K27me3_rep2; Homo sapiens; ChIP-Seq
HiPSCs were suspended in 1x Lysis buffer [15 mM NaCl, 60mM KCl, 5 mM MgCl2, 15 mM Tris-HCl (pH 7.5), 0.1 mM EGTA (pH 8.0), 0.3M Sucrose, 0.5mM DTT, 1% Nonidet P-40 (NP40), 1 mM PMSF, 1x Protease Inhibitor Cocktail (Nacalai Tesque)] and harvested with 1.2M Sucrose supplemented 1x Lysis buffer. Pellets were resuspended in micrococcal nuclease (MNase) buffer [0.3M Sucrose, 4mM MgCl2, 1mM CaCl2, 50mM Tris-HCl (pH 7.5)] and applied for chromatin fragmentation with 1.5U MNase for 15 min at 37°C. The reaction was stopped by adding 0.5M EDTA. The supernatant was added with Incubation buffer [50mM NaCl, 20mM Tris-HCl (pH 7.5), 0.1% NP40, 1mM PMSF] then incubated with Dynabeads M-280 Sheep anti-Mouse IgG (Invitrogen) and the following antibodies at 4°C overnight: mouse control IgG (#ab37355, Abcam), anti-H3K4me3 (#307-34813, Wako), anti-H3K27me3 (#301-95253, Wako). The ChIP samples were washed with the wash buffer [20mM Tris-HCl (pH 7.5), 10mM EDTA, 0.01% NP40, gradient salt concentration (75 - 100 - 175 mM NaCl)] 3 times. After 30 min RNase reaction at 37°C, DNA was eluted with Proteinase K (Nacalai Tesque) for 4 h at 56°C. To check the yield of chromatin shearing, 50% of input material was added with SDS (final concentration 5%) followed by RNase and Proteinase K reactions. DNA samples were purified using QIAquick PCR Purification Kit (Qiagen). ChIP samples were prepared as sequencing libraries using NEBNext Ultra II DNA Library Prep Kit for Illumina and sequenced using Illumina HiSeq 2000, and single-end reads were generated.