GSM5051761: M2_Input_1; Homo sapiens; ChIP-Seq
The ChIP experiment was performed as previously described (Lee et al., 2006) with minor modifications. Harvested iPSCs were crosslinked with 1% formaldehyde (methanol-free; Thermo Scientific) in PBS for 10 min at room temperature and quenched with 125 mM glycine. Crosslinked cells were incubated in lysis buffer 1 (50 mM HEPES-KOH pH 7.5, 1 mM EDTA, 140 mM NaCl, 10% glycerol, 0.5% NP-40, 0.25% TritonX-100, proteinase inhibitors) for 10 min at 4˚C. After centrifugation, the pellets were resuspended and incubated in lysis buffer 2 (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl, proteinase inhibitors) for 10 min at 4˚C. After centrifugation, the pellets were resuspended and incubated in lysis buffer 3 (50 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, proteinase inhibitors) for 10 min at 4˚C. Chromatin was sonicated using a Picoruptor (Diagenode) to achieve a mean DNA fragment size of around 200~400 base pairs. 1/10 volume of Triton X-100 was added and the supernatants (sonicated chromatin) after centrifugation were used for immunoprecipitation. Sonicated chromatin was diluted in lysis buffer 3 to an optimal volume for antibody reaction and pre-cleared for 35 min at 4˚C using Dynabeads M-280 Sheep anti-Mouse IgG beads (Invitrogen) or Dynabeads Protein A beads (Invitrogen) blocked with 0.5% BSA in PBS. Pre-cleared chromatin was then incubated with antibody that was pre-incubated with the appropriate Dynabeads in 0.5% BSA in PBS overnight at 4˚C. After the incubation for 6 h at 4˚C, the beads were washed 4 times in wash buffer 1 (20 mM Tris-HCl pH 8, 2 mM EDTA, 150 mM NaCl, 1% TritonX-100, 0.1% SDS), twice in wash buffer 2 (20 mM Tris-HCl pH 8, 2 mM EDTA, 500 mM NaCl, 1% TritonX-100, 0.1% SDS), and twice in wash buffer 3 (10 mM Tris-HCl pH 8, 1 mM EDTA, 250 mM LiCl, 1% Na-Deoxycholate, 1% NP-40). The washed beads were eluted in 10 mM Tris-HCl pH 8, 5 mM EDTA, 300 mM NaCl, 1% SDS, and crosslinks were reversed overnight at 65 ˚C. Input samples (chromatin equivalent of 2 × 10^5 cells) were treated in a similar manner. The following day, IP and Input samples were incubated with RNase A (Thermo Scientific) for 25 min at 37˚C, and Proteinase K (Thermo Scientific) for 40 min at 55˚C. IP or Input DNA was purified using the QIA quick PCR purification kit (QIAGEN). ChIP-seq libraries were prepared using a KAPA Hyper Prep Kit (Roche) following the manufacturer's guidelines. An adapter kit (FastGene) was used for the sample indexes. The average size and concentration of library samples were analyzed using LabChIP GX (PerkinElmer) and KAPA Library Quantification Kit (Roche), respectively.