ChIP-qPCR for H3K27me3 Occupancy

Cells were harvested by trypsin-EDTA and fixed with 1% formaldehyde at room temperature for 10 min. After quenching the formaldehyde with 125 mM glycine, the cells were lysed with mammalian cell lysis buffer (Pierce, Thermo Fisher Scientific, Inc.) and treated with micrococcal nuclease (Fermentas, Thermo Fisher Scientific, Inc.) at 37°C for 20 min. The fragmented DNA solution was further diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl and 167 mM NaCl) and pre-cleared by incubation with 10 μl Protein A Mag Sepharose (GE Healthcare) at room temperature for 2 hours. After the pre-clear step, 1 g of anti-H3K27me3 antibody or normal rabbit IgG was added to the cell lysate, and the mixture was incubated at 4°C overnight. After washing, protein/DNA complexes were eluted using elution buffer (1% SDS and 100 mM NaHCO₃). Reverse crosslinking of the protein/DNA complexes was performed by treating with 200 mM NaCl at 65°C for at least 5 hours, and the proteins were digested with proteinase K. The DNA was further purified using a Wizard^TM PCR Clean-Up kit (Promega, Madison, WI, USA). The H3K27me3 binding region in the promoter of ZEB1 or NEUROD1 was further detected using SYBR Green quantitative PCR method by the primer sets described by Chaffer CL et al. [20 (link)].