Pyromark q24 advanced pyrosequencing system

Bisulfite conversion of 500 ng of DNA was done with EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol, after which the genomic regions of the BACH2, MGAT3, IL6ST, LAMB1 and HNF1A genes were amplified using a PyroMark PCR kit (Qiagen). Amplified genomic regions were then sequenced using the PyroMark Q24 Advanced pyrosequencing system (Qiagen) and PyroMark Q24 Advanced CpG Reagents (Qiagen) to quantify methylation level at individual CpG dinucleotides. Assay sequences are listed in Supplementary Table S1, and assay maps are shown in Figures 4, 5 and 6. Sequences of PCR and pyrosequencing primers are listed in Supplementary Table S2.

We additionally examined whether high glucose affects methylation of CpG sites in regulatory regions of SERT, as suggested by our previous clinical study [37 (link)]. DNA methylation was quantified at 9 CpG sites in the promoter and 14 CpG sites in intron 1 of SERT (Table A2) by bisulfite pyrosequencing. Cellular DNA was isolated from ACH-3P cells using AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Bisulfite conversion was performed with 800 ng DNA per sample, using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA). Regions of interest were amplified using PyroMark PCR Kit (Qiagen, Hilden, Germany), and pyrosequencing was conducted on the PyroMark Q24 Advanced Pyrosequencing System with PyroMark Q24 Advanced CpG Reagents (both from Qiagen, Hilden, Germany), all following the manufactures’ recommendations. Primers used in the analysis are listed in Table A3. All assays included a negative control and a reference sample. Pyrosequencing quality control was performed using PyroMark Q24 Advanced Software (version 3.0.0, Qiagen, Hilden, Germany). The methylation levels of the CpG sites in each region were positively correlated, so the average methylation for the promoter and intron 1 region was used in the analyses.