Streptavidin coated beads

We also analyzed genomic DNA methylation in the MG and non-MG cells by pyrosequencing (Qiagen). PCR and sequencing primers were designed using PyroMark Assay Design 2.0 software (Qiagen). Pyrosequencing procedures were performed according to the manufacturer’s protocol. Regions of the ZNF350 promoter (region 1, -324 to -200 nt; region 2, -223 to -56 nt; region 3, -205 to +72 nt; and region 4, -17 to +72 nt from the transcription start site) were separately amplified by PCR using the primer sets shown in Supplementary Table 2. Bisulfite-converted genomic DNA (500 ng) was prepared using an EpiTect kit (Qiagen). The converted DNA was then amplified by PCR using a PyroMark PCR Master Mix kit (Qiagen). Then, the biotinylated PCR products were immobilized onto streptavidin-coated beads (GE Healthcare, Piscataway, NJ, USA), and the DNA strands were separated using the PyroMark denaturation solution (Qiagen). After washing and neutralization using a PyroMark Q24 Vacuum Workstation, the sequencing primer was annealed to the immobilized strand. DNA methylation was analyzed via highly quantitative bisulfite pyrosequencing with a PyroMark Q24 system (Qiagen). Data were analyzed using PyroMark Q24 software (Qiagen) to determine the methylation level (calculated as a percentage of methylation at each CpG).

DNA (500 ng) was subjected to bisulfite conversion with the EZ DNA methylation kit (Zymo Research) in accordance with the manufacturer’s instructions. Pyrosequencing was performed with the PyroMark Q96 ID system (Qiagen, Valencia, CA, USA) in accordance with the manufacturer’s recommendations. The PyroMark PCR Master Mix kit (Qiagen), streptavidin-coated beads (GE Healthcare, Uppsala, Sweden), PyroMark Gold Q96 reagents (Qiagen), PyroMark Q96 Vacuum Workstation, and PyroMark Q96 software (version 2.5.8, Qiagen) were used to determine and analyze DMSs.

Technical validation of the Infinium HumanMethylation450 BeadChip methylation data was performed by PyroSequencing (Qiagen) of three selected CpG sites (cg27483305, cg05688478, and cg13808071). Pre-designed PyroSequencing assays (PCR primers and sequencing primer) were used for the selected CpG sites (Qiagen) (Additional file 23). PyroSequencing was performed with the PyroMark™Q96 ID system (Qiagen) and all procedures were performed according to the manufacturer’s recommendations. In short, 500 ng genomic DNA from pancreatic islet of 50 human donors (32 males, 18 females) was bisulfite converted using EpiTect Bisulfite Kit (Qiagen), and 10 ng bisulfite converted DNA was then used as input for each PCR assay. Furthermore, PyroMark PCR Master Mix kit (Qiagen), streptavidin coated beads (GE Healthcare, Uppsala, Sweden), PyroMark Gold Q96 reagents (Qiagen) and PyroMark Q96 (version 2.5.8) software (Qiagen) were used for the analysis of DNA methylation, all according to the manufacturers’ recommendations.