Hiscript 2 reverse transcriptase reagent

One human immortalized epidermal cell line (HaCat) and three HNSC cell lines (M4E, CAL27 and TU686) were identified by STR. M4E, TU686 cells were cultured with RPMI1640 containing 10% fetal bovine serum (Gibco, Detroit, MI, USA) and penicillin (100 units/ml, Grand Island, NY, USA) and streptomycin (100 μg/ml, Grand Island, NY, USA). USA) and streptomycin (100 μg/ml, Grand Island, NY, USA) in RPMI1640 culture medium, HaCat, CAL27 cells were treated with 10% fetal bovine serum (Gibco, Detroit, MI, USA) and penicillin (100 units/ml, Grand Island, NY, USA) and streptomycin (100 μg/ml, Grand Island, NY, USA) in DMEM culture medium. Cells were incubated in a humidified environment in a constant temperature and humidity incubator at 37◦ C containing 5% CO2 and 95% air, and trypsin digested. Total RNA was extracted using FastPure Cell/Tissue Total RNA Extraction Kit (Vazyme, Nanjing, Jiangsu, China). Reverse transcription was performed according to the instructions of HiScript® II Reverse Transcriptase Reagent (Vazyme, Nanjing, Jiangsu, China). Quantitative polymerase chain reaction (Q-PCR) was performed with ChamQ Universal SYBR qPCR Master Mix reagent (Vazyme, Nanjing, Jiangsu, China) according to the instructions. GAPDH and U6 were used as internal reference genes. Relative expression was calculated using the ΔΔ-Ct method. The primer sequences are shown in Table S2.