BSAS of MT1G/MT1H promoter: HCC tissue
DNA methylation was assessed by next generation sequencing performing bisulfite amplicon sequencing (BSAS) of a region overlapping MT1G and MT1H promoter. The selected region was located at -598 bp/-999 bp from the transcription start site (TSS) of MT1G and it corresponded to the region -1151 bp/-750bp from the TSS of MT1H. DNA was bisulfite-converted using EZ DNA Methylation according to manufacturers protocol (Zymo Research, Irvine, CA, USA). The bisulfite converted region of interest was amplified by the high-fidelity DNA polymerase KOD -Multi & EPi (Toyobo, Osaka, Japan) using the following primers: Forward 5-TTTTGAGGAGTTGAATGGGTTTA-3, Reverse 5- AAATTCACACCAAACAATACCACTT -3. Library preparation was performed with an Illumina TruSeq Nano DNA library prep kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Libraries were quantified by qPCR using CFX96 Real Time System (BioRad, Hercules, CA, USA). Sequencing of the libraries was conducted on the Miseq system (Illumina) with 300 bp paired-end reads.