Pyromark q48 advanced reagents

DNA underwent bisulfite conversion utilizing the EZ DNA methylation kit (Zymo). The sequence for the CpG island preceding CIITApIV (chr16:10,972,877-10,973,234 from UCSC; available from https://genome.ucsc.edu/) was input into the PyroMark Assay Design software (QIAGEN, version 2.0.2.5) and the following primers were obtained from Sigma-Aldrich: CIITA Forward: 5′-GGGGGATGGAATATGTAAAATGTAG-3′, CIITA Reverse-Biotin: 5′-CCCCCAAACTCTAAACACAACAAACC-3′ and CIITA Sequencing: 5′-GGAATATGTAAAATGTAGGG-3′. PCR amplification of the region of interest was performed on bisulfite converted DNA using the PyroMark PCR kit (QIAGEN). Finally, pyrosequencing was performed in triplicate on a PyroMark Q48 sequencer using PyroMark Q48 Advanced Reagents (QIAGEN). Analysis of C/T ratio was performed by the PyroMark AutoPrep software.

For each sample, 1200 ng of gDNA was digested with 5 U each of HpaII (ER0512, Thermo Fisher Scientific) or MspI (ER0542, Thermo Fisher Scientific) in an end volume of 10 μl in 2× TangoY buffer (Thermo Fisher Scientific) for 4 hours at 37°C. After digestion, 5.5 μl of the sample was loaded on a PyroMark Q48 Autoprep machine (9002471, Qiagen) using PyroMark Q48 Advanced reagents (974002, Qiagen). The following nucleotide dispensation order was used: GTGTCACATGTGTG (85 (link)). The formula 1-[(HpaII(G)/EcoRI(T)/(MspI(G)/EcoRI(T))] × 100, where G and T represent nucleotides #9 and #8, respectively, was used to calculate % global methylation from the obtained pyrograms.