Validation of the sequencing data by bisulfite treatment

Bisulfite genomic sequencing is regarded as a gold-standard for detection of DNA methylation, because it provides a qualitative and quantitative method to apply to a limit number of loci [64]. Twenty-five DMRs were selected randomly to validate the reliability of the sequencing data using a nest PCR (nPCR). All the primers (Additional file 10) were designed using the website of Kismeth (http://katahdin.mssm.edu/kismeth/revpage.pl), and synthesized commercially (Invitrogen, Shanghai, China). Briefly, 1 μg of genomic DNA was treated by bisulfite according to the protocol of the EZ DNA Methylation-Gold Kit (Zymo Research, CA, USA), and used as the template for the following nPCR amplification. Then, the bisulfite treatment PCR (BS-PCR) was performed in a 50 μl reaction mixture, containing 25 μl premix EX Taq DNA polymerase (TaKaRa, Osaka, Japan), 25 μg bisulfite-treated DNA and 0.2 μM of each pair of primers with a nest PCR. Next, products were purified using Gel Extraction Kit (Axygen, CA, USA), and cloned into the pMD19-T Simple Vector (TaKaRa, Osaka, Japan). Each DMR amplified with 10–14 clones was sequenced by Invitrogen Trading Shanghai Co.Ltd. (Shanghai, China), and the sequencing results were spliced and edited by BioEdit and vector NTI8 software, then analyzed in the Kismeth website.