Chromatin immunoprecipitation

Approximately 1.0 × 10⁷ proliferating SCs for each antibody were fixed with 1% formaldehyde at room temperature for 10 minutes. The cell lysates were sonicated with a Covaris S2 sonicator to shear DNA. Dynabeads Protein A (Invitrogen, Carlsbad, CA) conjugated with 10 μg of each primary antibody was added, followed by incubation at 4°C overnight. The beads were washed 5 times with RIPA buffer (0.2% NP-40, 0.2% Na-deoxycholate, 0.16 M LiCl, 10 mM EDTA, 20 mM HEPES-KOH, pH 7.6) and eluted with elution buffer (1% SDS, 50 mM EDTA, 100 mM Tris-HCl, pH 8.0). The eluate was incubated at 65°C overnight to reverse the crosslinking, followed by incubation at 55°C for 1 hour in the presence of proteinase K. DNA was purified using a MinElute PCR Purification Kit (Qiagen, Hilden, Germany) and quantified by real-time PCR (Thermal Cycler Dice Real Time System II (Takara Bio, Japan)). All primer sequences are listed in S1 Table.