Bisulfite sequencing (BSP) and next-generation sequencing (NGS) 13

Genomic DNA was extracted from ESCC or non-tumor tissue using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. DNA concentration and quality were determined using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The samples were subjected to bisulfite treatment with the ZYMO EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA) according to the manufacturer's protocol. The promoter region of each target gene - i.e., the sequence 2000 bp upstream of the transcription start site—was obtained by searching the NCBI database. Methyl Primer Express v1.0 software (Thermo Fisher Scientific) was used to identify and predict CpG sites in the promoter sequence and primers were designed for PCR amplification of this region. The amplified product, which was about 218 bp in size, was purified using magnetic beads for sequencing library construction (Life Technologies, Carlsbad, CA, USA), and a barcode was added for second-generation sequencing on the Iontorrent PGM platform (Life Technologies). If the sequencing result of the OCTs' promoter region after BSP treatment is C, it indicates that the CpG site was methylated; if the sequence of the OCTs promoter region sequencing after BSP treatment was T, it indicated that the methylation of the CpG site was not occurred. The methylation rate of OCT gene promoter sequences was calculated as the number of methylated CpG sites divided by the total number of CpG sites. The forward and reverse primers used for BSP and NGS experiments were as follows: Oct1/POU2F1, 5'-ATTGAGGGYGTTGTTTTAGTT-3' and 5'-CCTCAAAAAAACTCCACC-3'; Oct4/POU5F1, 5'-GTGGTTAGGTATTTTGGGAGGT-3' and 5'-CAAACTAAACTCR AACTCCC-3'; Oct6/POU3F1, 5'-TYGAGATTTTTTTTTTTTGGAATT-3' and 5'-AACRA TTCTACAATCCTACRC-3'; and Oct11/POU2F3, 5'-TTGTAATTTTAGGGAAG TTTAATTGA-3' and 5'-CTCAAATTCTCTTATCCCTAATTAAA-3' (where R = A or G; Y = C or T).

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