GSM5597326: H3K27me3 ChIP-seq Lin- WT Rep2; Mus musculus; ChIP-Seq
ChIP experiments were performed as previously described with minor modifications (Kadota and Nagata, 2014). Briefly, to perform H3K27me3 and H3K4me3 ChIP, mouse bone marrow Lin- cells (~3x106) were crosslinked with 1% formaldehyde for 10 min at RT. The reaction was stopped by adding glycine (final concentration, 125 mM) for 5 min at RT. Crosslinked cells were treated with hypotonic buffer (10 mM HEPES-NaOH, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 % NP-40, 1 mM DTT, 1 mM PMSF, 1x protease inhibitor cocktail (Sigma, P8340)) at 4ºC for 10 min and collected in a tube. The cells were lysed in 150 ml of ChIP lysis buffer (50 mM Tris-HCl, pH 7.9, 10 mM EDTA, 1% SDS, 1 mM PMSF, protease inhibitor cocktail (Sigma, P8340)) on ice for 10 min, and sonicated for 35 cycles (30'' ON + 30'' OFF) using Diagenode Bioruptor to shear the chromatin to 200-1,000 bp fragments. The lysate was diluted with 9x volumes of ChIP dilution buffer (16.7 mM Tris-HCl, pH 7.9, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 1 mM PMSF). The fragmented chromatin was pre-cleared using 15 ml of pre-washed Protein A/G agarose beads (Thermo Fisher, 20423) for 1 hour at 4ºC. The pre-cleared chromatin fragments were equally separated into two aliquots. Each aliquot was incubated with either 2 ml of anti-H3K27me3 (mAbcam6002; Abcam) or 2 ml of anti-H3K4me3 (ab8580; Abcam) antibodies at 4ºC overnight with rotation, followed by incubation with pre-washed Protein A/G agarose beads (Thermo Scientific, 20423) for 1-2 hours at 4ºC. DNA-protein complexes attached to agarose beads were washed at 4ºC with the following buffers: low salt wash buffer (20 mM Tris-HCl, pH 7.9, 100 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) for 10 min, high salt wash buffer (20 mM Tris-HCl, pH 7.9, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) for 10 min, the LiCl buffer (10 mM Tris-HCl, pH 7.9, 250 mM LiCl, 1% NP-40, 1% deoxycholate acid, 1 mM EDTA) for 10 min, and twice with TE buffer for 5 min. DNA-protein complexes were eluted by incubating the beads in elution buffer (1% SDS, 0.1 M NaHCO3) for 15 min at RT. Reverse crosslinking was performed by incubating samples in 200 mM of NaCl at 65ºC overnight followed by Proteinase K (20 mg/ml) treatment for 2 hrs at 55ºC. DNA was purified using MiniElute PCR purification kit (QIAGEN, 28006). To perform YY1 ChIP, bone marrow Lin- cells (~1.5x106) were crosslinked with 1% formaldehyde for 15 min at RT and the reaction was stopped by adding glycine (final concentration, 125 mM) for 5 min at RT. Crosslinked cells were treated with cell lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 1 mM DTT, 1 mM PMSF, 1x protease inhibitor cocktail (Sigma, P8340)) for 10 min at 4ºC. To digest chromatin into <1,000 bp fragments, the pellet was washed twice with MNase reaction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 0.2% NP-40, 1 mM DTT, 1 mM PMSF, 1x protease inhibitor cocktail), resuspended in 200 ml of MNase reaction buffer containing 2.5U of MNase (NEB, M0247) and incubated for 10 min at 37ºC. MNase digestion was stopped by adding 20 ml of 0.5M EDTA into the reaction. Nuclear membrane was broken up by sonicating the lysate for 3 cycles (output level 6, 10'' ON + 20'' OFF) using Microson ultrasonic cell disruptor (Misonix). Lysate was spun at 21,000xg for 10 min at 4ºC, and the supernatant containing fragmented chromatin was incubated with 2 ml of anti-YY1 antibody (D5D92Z, Cell Signaling, 46395) overnight at 4ºC. DNA-protein complexes were pulled down with addition of 10 ml of pre-washed Protein G Magnetic Beads (Invitrogen, 10003D) and incubation for 1-2 hours at 4ºC. DNA-protein complexes were eluted in elution buffer (1% SDS, 0.1 M NaHCO3) by incubating at 65ºC for 40 min, and reverse crosslinked as described above. All ChIP DNA samples were purified using MinElute PCR purification kit (Qiagen,28006) and quantified using the fluorometric quantitation Qubit 2.0 system (Thermo Fisher Scientific). ChIP-seq libraries were prepared using KAPA HyperPrep kit (KAPA, 07962347001). Libraries were sequenced on Illumina HiSeq 4000 (single-end; H3K27me3, and H3K4me3 ChIP-seq) and on Illumina NovaSeq 6000 (paired-end; YY1 ChIP-seq).