GSM5572054: GSCLC_H2Aub_2; Mus musculus; ChIP-Seq
The ChIP-seq library preparation was performed as previously described (Lee et al., 2006) with minor modifications. We used harvested mESCs and EpiLCs, and FACs-sorted BV-positive cells for d2 mPGCLCs and d4c7 mPGCLCs samples, and FACS-sorted AAG-positive cells for GSCs and GSCLCs samples. Briefly, the harvested cells were crosslinked with 1% formaldehyde (Thermo Fisher Scientific)/PBS for 10 min at RT and quenched with 125 mM glycine. Crosslinked cells were lysed consecutively using LB1 (50 mM HEPES-KOH pH 7.5, 1 mM EDTA, 140 mM NaCl, 10% glycerol, 0.5% NP-40, 0.25% Triton-100, protease inhibitors (Roche, 11873580001)), LB2 (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl, protease inhibitors), and LB3 (50 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, protease inhibitors) and then sonicated by picoruptor to achieve a mean DNA fragment size of around 200-400 bp. Sonicated chromatin was incubated with  Dynabeads M-280 Sheep anti Mouse IgG beads (Thermo Fisher Scientific) or Dynabeads ProteinA beads (Thermo Fisher Scientific) for 35 min at 4°C for preclear. Precleared chromatin was then incubated with antibody that was preincubated with the appropriate Dynabeads in 0.5% BSA (Gibco) in PBS as follows: chromatin equivalent of 5 x 105 cells with anti-H3K4me1 (rabbit monoclonal, CST, D1A9, 5 μl),  anti-H3K9me2 (mouse monoclonal, MBL, MABI0317, 5 μl), anti-H3K27me3 (mouse monoclonal, MBL, MABI0323, 5 μl); 1 x 106 cells with anti-H3K4me3 (mouse monoclonal, MBL, MABI0304, 5 μl), anti-H3K9me3 (mouse monoclonal, MBL, MABI0318, 5 μl), anti-H3K36me2 (rabbit monoclonal, CST, C75H12, 5 μl), anti-H2AK119ub1 (rabbit monoclonal, D27C4, 10 μl), anti-H3K36me3 (rabbit polyclonal, Active Motif, 61101, 2 μl); 1.5 x 106 cells with anti-H3K27ac (mouse monoclonal, MBL, MABI0309, 5 μl); 2 x 106 cells with anti-CTCF (rabbit monoclonal, CST, D31H2, 5 μl), anti-Laminb1 (rabbit polyclonal, Proteintech, 12987-1-AP, 10 μl); 3 x 106 cells with anti-Setdb1 (rabbit ployclonal, Proteintech, 11231-1-AP, 10 μl); 4 x 106 cells with anti-Ring1b (rabbit monoclonal, CST, D22F2, 10 μl); and 4.5 x 106 cells with anti-Rad21 (rabbit monoclonal, ab992, 5 μl). After incubation for 6 h at 4°C, the beads were washed 4 times in wash buffer 1 (20 mM Tris-HCl pH8.0, 2 mM EDTA, 150 mM NaCl, 1% TritonX-100, 0.1% SDS), 2 times in wash buffer 2 (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% TritonX-100, 0.1% SDS), and 2 times in wash buffer 3 (10 mM Tris-HCl  pH 8.0, 1 mM EDTA, 250 mM LiCl, 1% Na-Deoxycolate, 1% NP-40). Then, the washed beads were eluted in 10 mM Tris-HCl pH 8.0, 5 mM EDTA, 300 mM NaCl, 1% SDS, and crosslinks were reversed overnight at 65°C. Input samples were treated in a similar manner. The following day, IP and Input samples were incubated with RNaseA (Thermo Fisher Scientific) and proteinase K (Thermo Fisher Scientific). IP or Input DNA was purified using QIA quick PCR purification kit (QIAGEN). ChIP-seq libraries were prepared using KAPA Hyper Prep Kit (KAPA) following the manufacturer's guidelines. An adaptor kit (Fastgene, FG-NGSAD24) was used for the sample indexes. The average size and concentration of libraries were analyzed using LabChIP GX (PerkinElmer) and KAPA library Quantification kit (KAPA), respectively.