For qRT-PCR, total RNA was prepared using TRIzol reagent (Thermo Fisher Scientific Cat#15596026), and 1 μg of RNA was reversely transcribed with random primer (Thermo Fisher Scientific Cat#48190011), dNTP mix (Thermo Fisher Scientific, Cat#18427013), and M-MuLV Reverse Transcriptase (New England Biolabs, Cat#M0253L). The levels of mRNA were qualitatively measured using a SYBRGreen supermix (Bio-Rad, Cat#1708880). GAPDH was used as an internal control.
ChIP-qPCR assays were performed as previously described (Ding et al, 2013 (link)). Briefly, SCC cells were sequentially treated with dimethyl 3,3’-dithiobispropionimidate-HCl (DTBP; Cat#20665, Thermo Fisher Scientific) solution and formaldehyde, and harvested with a cell scraper. The cell pellet was lysed with ChIP lysis buffer and sonicated to generate 200–500 bp DNA fragments with a sonicator. The fragmented chromatins were immunoprecipitated with anti-BMI1 (Cell Signaling Technology, Cat#6964), anti-Ubiquityl-Histone H2A (Lys119) (Cell Signaling Technology, Cat#8240) overnight at 4°C. The precipitated DNA-chromatin products were purified with ChIP DNA clean & concentrator kit (Cat#D5205, Zymo Research) and the DNA levels were quantified by qPCR. Data is presented as the percentage of input DNA. The primer sequences used for qRT-PCR and ChIP-qPCR were listed inTable S1 .
ChIP-qPCR assays were performed as previously described (Ding et al, 2013 (link)). Briefly, SCC cells were sequentially treated with dimethyl 3,3’-dithiobispropionimidate-HCl (DTBP; Cat#20665, Thermo Fisher Scientific) solution and formaldehyde, and harvested with a cell scraper. The cell pellet was lysed with ChIP lysis buffer and sonicated to generate 200–500 bp DNA fragments with a sonicator. The fragmented chromatins were immunoprecipitated with anti-BMI1 (Cell Signaling Technology, Cat#6964), anti-Ubiquityl-Histone H2A (Lys119) (Cell Signaling Technology, Cat#8240) overnight at 4°C. The precipitated DNA-chromatin products were purified with ChIP DNA clean & concentrator kit (Cat#D5205, Zymo Research) and the DNA levels were quantified by qPCR. Data is presented as the percentage of input DNA. The primer sequences used for qRT-PCR and ChIP-qPCR were listed in