GSM2166075: REH H3K4me3 [ChIP-Seq]; Homo sapiens; ChIP-Seq
40 million cells were crosslinked with 1% formaldehyde for 10-15 mins.  The reactions were quenched by adding glycine to a final concentration of 125 mM, and the cells were centrifuged and washed twice with ice-cold PBS. For each ChIP 5 million cells was used. Nuclei was extracted by washing cell pellet twice with 1 ml of MNase buffer (10mM Tris ph 7,4, 10mM NaCl, 5mM MgCl2, 0,5% IGEPAL CA-630 [Sigma], 1x protease inhibitor cocktail [PIC, Roche], 1 mM PMSF [Thermofisher]). Nuclei were spun down (1500 x g, +4 °C, 5 min) and suspended into 90µl of MNase buffer supplemented with 5mM CaCl2 and 0.1% Triton-X. Different amounts of MNase (0.5-20U; #88216, Thermofisher) was added to the nuclei in 10 µl volume and incubated at 37°C for 10 mins.  To stop the reaction, 100 µl of 2xLysis buffer was added to the reaction (1% SDS, 40mM EDTA, 100mM Tris-HCl pH 8.1) and samples were sonicated using Bioruptor (Diagenode) for 5 cycles (30 s – 30 s) to break the nuclei.  The lysate was cleared by centrifugation and supernatant was diluted 5 x with dilution buffer (for H3K4me3; 20mM Trix-HCl pH 7.4, 100mM NaCl, 2mM EDTA, 0.5% TritonX, PIC). The diluted lysate was pre-cleared by rotating for 2 h at 4°C with 60 µl 80% CL-4B sepharose slurry (GE Healthcare; Before use, sepharose was washed twice with TE buffer, blocked for 1h min at room temperature with 0.5% BSA and 20 µg/ml glycogen in 1 ml TE buffer, washed twice with TE and brought up to the original volume with TE). The beads were discarded, and 1% of the supernatant were kept as ChIP input. The protein of interest was immunoprecipitated by rotating the supernatant with 3-5 µg antibody overnight at 4°C. Antibody H3K4me3 (ab8580) was purchased from Abcam. The Ab was captured using 25 µl blocked Protein G Sepharose® 4 Fast Flow (GE Healthcare, sepharose was blocked rorating overnight at 4°C) and rotating the sample for 2 h at 4°C. The beads were pelleted (1 min, 1000× g, 4°C), the supernatant discarded, and were washed three times with wash buffer I (20 mM Tris/HCl pH 7.4@20°C, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 2 mM EDTA), twice with buffer II (20 mM Tris/HCl pH 7.4@20°C, 500 mM NaCl, 1% Triton X-100, 2 mM EDTA) and buffer III (10 mM Tris/HCl pH 7.4@20°C, 250 mM LiCl, 1% IGEPAL CA-630, 1% Na-deoxycholate, 1 mM EDTA), once with TE+0.2% TritonX and twice with TE. Immunoprecipitated chromatin was eluted twice with 100 µl elution buffer (TE, 1% SDS). The NaCl concentration was adjusted to 300 mM with 5 M NaCl and crosslinks were reversed overnight at 65°C. The samples were sequentially incubated at 37°C for 2 h each with 0.33 mg/ml RNase A and 0.5 mg/ml proteinase K. The DNA was isolated using the ChIP DNA Clean & Concentrator (Zymo Research) according to the manufacturer’s instructions. Sequencing libraries were prepared from collected DNA by blunting, A-tailing, adaptor ligation as previously described (Heinz 2010; MolCell) using barcoded adapters (NextFlex, Bioo Scientific). Between the reactions, the DNA was purified using Sera-Mag SpeedBeads (Thermo Scientific). Libraries were PCR-amplified for 15-16 cycles and size selected for 230-350bp fragments by gel extraction.