Champagne taq dna polymerase

Genomic DNAs of human UBC tissues, adjacent non-tumorous tissues, and cell lines were isolated. The DNA was performed bisulfite conversion through The EpiMark Bisulfite Conversion Kit (Cat# E3318S, New England Biolabs). The bisulfite-converted DNA was immediately amplified with Champagne Taq DNA Polymerase (Cat# P122-d1, Vazyme, China). PCR products were cloned into pMD-19T (Cat #3271, TaKaRa, China) vector and processed to sequencing. The sequencing results were analyzed with BiQ Analyzer^{44 (link)}. The gray level of each point represented the CpG island methylation level and was calculated by number of T vector clones with CG clones/(TG clones + CG clones). The primers of bisulfite sequencing amplification were described in Table S5.

The one-step RT-qPCR assay (referred to as our RT-qPCR assay) was performed in 96-well plates with a reaction volume of 30 μL. For the assay, 3 μL of 10× Taq Buffer, 0.24 μL of Champagne Taq DNA Polymerase, 0.35 μL of HiScript II Reverse Transcriptase, 0.25 μL of Murine RNase inhibitor, 0.3 μL of heat-labile UDG, 2.4 μL of MgCl2 and 0.6 μL of dNTP mix (Vazyme Biotech Co., Ltd., Nanjing, China), 0.6 μL of forward primers and 0.6 μL of reverse primers for each gene, 0.15 μL of probes for each gene, 13.81 μL of nuclease-free water, and 5 μL of template were mixed per reaction. The cycling conditions for each assay consisted of incubating the template at 55 °C for 5 min and 95 °C for 30 s, followed by 45 cycles of RT-qPCR, with each cycle consisting of 95 °C for 5 s and then 62 °C for 20 s. All reactions were carried out with a SLAN^®-96S Real-Time PCR System (Hongshi Medical Technology Co., Ltd., Shanghai, China).