Vacuum prep tool

Pdx1 promoter methylation status results obtained by BSP were confirmed by pyrosequencing. Sodium bisulfite modification of genomic DNA of 7.5 × 10⁴ cells was carried out as described above. Converted DNA was eluted in 15 μL and 2 μL for each PCR cycle. The primers used for PCR and sequencing were designed using PyroMark assay design software, version 2.0.01.15 (Qiagen, Hilden, Germany). The pyrosequencing primers are shown in Table S1. These primers were designed to hybridize with CpG-free sites to ensure methylation-independent amplification. PCR was performed with biotinylated primers to convert the PCR product to single-stranded DNA templates, using the Vacuum Prep Tool (Biotage, Uppsala, Sweden), according to the manufacturer’s instructions. Pyrosequencing reactions and methylation quantification were performed using a PyroMark Q24 System, version 2.0.6 (Qiagen).

DNA from the MLST alleles IGS1, GPD1 and URA5 were PCR amplified using biotinylated primer pairs targeting the region containing the SNP distinguishing the ST5 genotype. PCR amplifications were performed in 60 μl reactions containing 1 × Hotstart PCR buffer, 1.5 mM of MgCl2, 200 μM of dNTP, 10 pM of each primer, 1.25 Units of Hotstart DNA polymerase (Qiagen, USA) and 3 μl of template DNA. Reactions were cycled once at 95°C for 15 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 1 min, with a final elongation of 72°C for 10 min. All PCR amplifications were visualized on 2% agarose gels prior to pyrosequencing. A pyromark Q96 ID DNA pyrosequencer (Biotage, Sweden) was used to detect the SNPs of interest in each allele as per the manufacturer’s recommendations. PCR amplicons were combined with 56 μl of binding buffer and 4 μl of streptavidin sepharose beads. The resulting mixture was agitated for 5 min before denaturation in denaturation buffer and washing with the Vacuum Prep Tool (Biotage, Sweden). DNA fragments were transferred into a 96-well plate containing 3.5 pmol of sequencing primer in 40 μl of annealing buffer and the DNA sequencing reaction was performed using the Pyro Gold Kit (Biotage, Sweden). The SNP detection mode in the software PyromarkID v1.0 (Biotage, Sweden) was used to analyze sequence data.