Top10 cells

PCR reactions (25 μL) contained 0.4 mM forward and reverse primers (Table 2), 12.5 μL Taq PCR master mix 2 × (Sangon Biotech), and 20–25 ng of genomic DNA. The PCR cycle conditions are given in Table 3. After agarose gel electrophoresis and ethidium bromide staining, PCR amplicons were visualized with a Gel Doc XR+ system (Bio-Rad). PCR products were purified from the gel using a universal DNA purification kit (Tiangen Biotech), then ligated into the pUCm-T vector (Sangon Biotech). Recombinant plasmids were transformed into TOP10 cells (Tiangen Biotech), which were streaked on agar plates containing 50 μg/mL ampicillin and grown overnight at 37°C. Three positive colonies were selected for Sanger sequencing (Sangon Biotech).

Genomic DNA was extracted via proteinase K digestion and phenol/chloroform extraction [25 (link)], denatured with NaOH and modified using sodium bisulfite and hydroquinone. Bisulfite-treated DNA was purified using a Wizard DNA clean-up system (Promega, Madison, WI, USA), following the manufacturer’s instructions. DNA was precipitated with ethanol after treatment with NaOH and eluted into 50 μl distilled water. The final products were stored at −80°C until use.
Bisulfite treatment was performed using an EpiTect Bisulfite Kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual. Bisulfite-converted DNA was amplified using nested PCR. Primer sequences are shown in Additional file 1. Each 25 μl PCR reaction mixture contained 4 μl of bisulfite-treated DNA, and reactions were performed according to the method described by Kagamiet al. [26 (link),27 (link)]. The presence of amplified products was confirmed by electrophoresis on a 1.5% agarose gel.
PCR products were retrieved and ligated into the pMD19-T Vector System (TaKaRa). After transformation via heat shock into 200 μl competent Top10 cells (Tiangen, Beijing, China), colonies were isolated and cultured. For each sample, 15 positive clones were sequenced.