Ab213257

ChIP was performed using EZ-Magna ChIP A/G Chromatin Immunoprecipitation kits (17-371, Millipore, Billerica, MA, USA). Briefly, the cells were sonicated and centrifuged at 12,000 g for 10 min at 4 °C to remove insoluble precipitates. The cells were subsequently incubated with protein G agarose beads at 4 °C for 1 h, and centrifuged at 5000 g for a total of 1 min. Next, 10 μL (1%) of the supernatant was collected as the 'Input', while the remaining supernatant was divided into 2 portions and incubated with antibodies against H3K9me3 (ab8898, dilution ratio of 1:20, Abcam Inc., Cambridge, UK), H3K9me2 (ab1220, dilution ratio of 1:10, Abcam Inc., Cambridge, UK), H3 (ab213257, dilution ratio of 1: 5, Abcam Inc., Cambridge, UK), and NC rabbit antihuman IgG (ab2410, dilution ratio of 1:25, Abcam Inc., Cambridge, UK) at 4 °C overnight. The precipitated protein-DNA complex was then incubated with protein G agarose beads for 1 h at 4 °C. After centrifugation at 5000 g for 1 min, the supernatant was discarded. The protein-DNA complex was fragmented overnight at 65 °C. The DNA fragment was recovered and used as an amplification template for RT-qPCR. The primers employed to detect the quantity of HES1 promoter were agomir NC (5′-UUCUCCGAACGUGUCACGUTT-3′) and miR-216b agomir (5′-AAAUCUCUGCAGGCAAAU GUGA-3′).