Pyromark q24 software version

DNA was isolated from the hippocampus tissue using the classic phenol–chloroform method. Briefly, the tissue sample was incubated in lysis buffer (100 mM Tris–HCl pH 7.5, 20 mM EDTA, 165 mM NaCl, 1.0% SDS, 70 μg/ml proteinase K) at 55 °C overnight. The resulting lysate was treated with an equal volume of a phenol/chloroform/isoamyl alcohol mixture (Nacalai, Japan) and purified through ethanol precipitation. The concentration of the extracted DNA was measured using the NanoDrop ND-1000 Spectrophotometer.
Subsequently, 1 μg of DNA was subjected to bisulfite conversion using the EZ DNA Methylation-Gold Kit (Zymo Research, USA) following the manufacturer's protocols. The bisulfite-treated DNA was then used for PCR amplification using the PyroMark PCR kit (Qiagen, USA) according to the manufacturer's protocols. The pyrosequencing reaction was performed on a PyroMark Q24 instrument (Qiagen, USA). The resulting data were analyzed and quantified using the PyroMark Q24 software version 2.0.8 (Qiagen, USA). The PCR and pyrosequencing primers used were listed in Table S1, designed with the PyroMark Assay Design software v2.0.2.5 (Qiagen, USA).

The gDNA samples were bisulfite-converted using the EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The bisulfite-converted DNA (bisDNA) samples were diluted to 20 ng/µL. The polymerase chain reaction (PCR) of the diluted bisDNA samples was performed with the PyroMark^® Kit (Qiagen, Hilden, Germany). According to our established protocol, there was an initial activation period of 15 min. at 95 °C, a 3-step cycle of denaturation (94 °C for 30 s), annealing (58 °C for 5 s), and extension (72 °C for 15 s) for 45 cycles. The PCR process was terminated with a final extension period of 72 °C for 10 min. PyroMark assays (biomers.net, Ulm, Germany), primer sequences, and the sequence to analyze, based on the work by Hunter et al. 2019 [15 (link)], are listed in Table 2.
Gel electrophoresis was performed on a 2% agarose gel using GeneRuler 100 bp DNA Ladder (ThermoFisher Scientific, Waltham, MA, USA) to check the PCR products. CpG methylation was determined using PyroMark Q24 (Qiagen, Hilden, Germany) and PyroMark Gold Q24 Reagents (Qiagen, Hilden, Germany). The nucleotide dispensing order was generated by entering the sequence to be analyzed into the PyroMark Q24 software version 2.0.8 (Qiagen, Hilden, Germany). The methylation at each CpG site was evaluated using the PyroMark Q24 software in the CpG mode.