Pyromark gold q96 sqa reagents

DNA extraction and sodium bisulfite treatment were performed in the same manner as above for WGBS sample processing. Bisulfite pyrosequencing primers were designed using PyroMark Assay Design software (Qiagen, Hilden, Germany) to target CpGs within DMRs from the male replication comparison. Bisulfite-converted DNA from each sample was amplified in triplicate for each primer set using the PyroMark PCR kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, except using 50 ng of bisulfite-converted DNA and 40 PCR cycles. Each PCR product was checked with gel electrophoresis for specific amplification. Pyrosequencing was conducted using PyroMark Gold Q96 SQA Reagents (Qiagen, Hilden, Germany) with the PyroMark Q96 instrument (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Mean percent methylation for each region was compared to diagnosis outcome using linear regression and compared to WGBS methylation using Pearson’s r.

ATF2 knockdown was performed as described and DNA/RNA was extracted using the DNeasy/RNeasy kits (Qiagen, Crawley, UK), respectively. Total RNA was reverse transcribed using the Quantitect RT kit (Qiagen, Crawley, UK). Pyrosequencing primers were designed using Pyromark Assay Design 2.0 software (Qiagen, Crawley, UK) to measure the DNA methylation levels of ESR1 and PGR after ATF2 knockdown and synthesised by Eurofins MWG Operon (Ebersberg, Germany). The primer sequences are listed in Supplementary Material and Methods, Table 3. Genomic DNA was treated with sodium bisulphite using the EZ DNA methylation Kit (Zymo Research, CA, USA). PCR amplifications were performed in a final volume of 25 μl using HotStarTaq Master Mix (Qiagen, Crawley, UK), 200 nM biotinylated primer, 400 nM non-biotinylated primer and 60 ng of bisulfite-treated genomic DNA. The thermal profile was 95 °C for 5 min followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 51–56 °C for 30 s and extension at 72 °C for 30 s. The PyroMark Gold Q96 SQA Reagents and the PyroMark Q96 ID instrument (Qiagen, Crawley, UK) were used for pyrosequencing analysis following the supplier’s protocol. The methylation index for each promoter was calculated as the mean value of mC/(mC + C), where C is unmethylated cytosine and mC is 50′ methyl-cytosine, for all examined CpGs in the target sequence.