ChIP-qPCR for DNMT1 binding

Cells were fixed with 1% formaldehyde at room temperature for 10 min to crosslink DNA and proteins. After crosslinking, the crosslinked DNA and proteins were randomly fragmented by ultrasonic treatment, and the supernatant was collected into three tubes. Next, anti-RNA polymerase II antibody (positive control), normal mouse IgG (NC) (ab109489, 1:100, Abcam, Cambridge, MA, USA), and an antibody specific for the target protein mouse anti-DNMT1 (ab183403, 1:50, Abcam, Cambridge, MA, USA) were added to the cells in the three tubes and incubated overnight at 4 °C. On the following day, the endogenous DNA–protein complexes were precipitated by using Protein A/G-Sepharose and briefly centrifuged to discard the supernatant. The nonspecific complexes were washed, decrosslinked at 65 °C overnight, and purified by phenol/chloroform to extract the DNA fragments. Finally, the binding of DNMT1 to the HOXA11 promoter region was detected by using primers specific for the HOXA11 gene promoter region^{21 (link)}.