Pyromark assay design 2

The PyroMark Assay Design 2.0 software (Biotage, Uppsala, Sweden) was used for design of PCR and sequencing primers (Additional file 1: Table S1). Assays were established using the EpiTect PCR Control DNA set (Qiagen) with 0, 25, 50, 75, and 100% methylation. PCR reactions were performed in a total volume of 25 μl using the FastStart Taq DNA polymerase system (Roche Diagnostics, Mannheim, Germany). The 25 μl reaction consisted of 2.5 μl 10× PCR buffer, 20 mM MgCl₂, 1.0 μl dNTP (10 mM) mix, 10 pmol of forward and reverse primer, 1 IU of FastStart polymerase, 1 μl (approximately 100 ng) bisulfite converted template DNA, and 18.3 μl PCR-grade water. For SLC17A4, 2.0 μl template DNA and 17.3 μl water were used.
To reduce technical noise (batch effects), bisulfite conversion and PCR (of D-GDM, I-GDM, and control samples) were performed simultaneously in 96-well microtiter plates. Pyrosequencing was performed on a PyroMark Q96 MD system (Qiagen) using the PyroMark Gold Q96 CDT reagent kit (Qiagen), 10 pmol of sequencing primer, and Pyro Q-CpG software (Qiagen). In our experience, the average methylation difference between technical replicates (including bisulfite conversion, PCR, and pyrosequencing) is approximately 1–2 percentage points. Artificially methylated and unmethylated DNA standards (Qiagen) were included as controls in each pyrosequencing run.

DNA was isolated using TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China). DNA bisulfite conversion was modified using EZ DNA CT conversion reagent (Zymo Research Corporation, Irvine, CA, USA) following the manufacturer’s protocol. The primers for Fyn bisulfite DNA PCR and sequencing were designed using Pyromark Assay Design 2.0 software (Biotage, Uppsala, Sweden) and listed in Additional file 1. Gene model of Fyn was presented as Additional file 1: Fig. S1.
Bisulfite DNA PCR was performed in a total volume of 25 μl using the TaKaRa EpiTaq HS system (TaKaRa, Dalian, China). Pyrosequencing was performed on a PyroMark Q24 MD system (Qiagen) using the PyroMark Gold Q24 reagent kit (Qiagen) with 10 pmol of sequencing primer. Data were analyzed using Pyro Q-CpG software (Qiagen).