Cpgenome turbo bisulfite modification kit

To validate the BS-Seq results of the two samples, bisulfate PCR was performed. First, 1 μg of genomic DNA was treated with bisulfite using a CpGenome Turbo Bisulfite Modification Kit (Millipore Corp, Billerica, MA, USA). Primers for BS-Seq were designed using the Meth-Primer Program [50 (link)]. The PCR products were purified and cloned into pEASY-Blunt Zero Cloning Vector (TransGen Biotech, Beijing, China) for sequencing. In total, 20 clones were sequenced for each PCR product. The BS-Seq results were analyzed using the BiQ Analyzer software [51 (link)].

The Bisulfite Sequencing PCR (BSP) protocol was carried out as previously described (23 (link)). Using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China), the DNA of SGC7901 and BGC823 cells were extracted. The DNA was modified using CpGenome™ Turbo Bisulfite Modification Kit (Millipore, USA). The differentially methylated regions (DMRs) of H19 were amplified using nested PCR. The products, which included 10 positive clones, were analyzed using the BiQ Analyzer software (http://biq-analyzer.bioinf.mpi-inf.mpg.de/tools/MethylationDiagrams/index.php). The primers of H19 DMR are listed in Table S3.

Genomic DNA was extracted from cells of indicated groups using the standard phenol–chloroform extraction method. Genomic DNA was treated with bisulfite using CpGenome Turbo Bisulfite Modification Kit (Millipore, Billerica, MA, USA) according to the manufacturer’s manual. The modified DNA was amplified using Platinum Taq DNA polymerase (Life Technologies) with the respective primer sets that recognize bisulfite-modified DNA only (primer sequences listed in Supplementary Table S6). Then the PCR products were cloned into pMD 18-T vector (Takara, Shiga, Japan), followed by Sanger sequencing.

Bisulfite conversion of the isolated genomic DNA was performed by CpGenome Turbo Bisulfite Modification Kit (Millipore, Billerica, MA) according to the manufacturer’s instructions. Bisulfite-treated DNA was amplified by PCR using Quick Taq HS DyeMix (Toyobo, Osaka, Japan). All primer sequences are listed in S1 Table. PCR products were cloned into T-Vector pMD20 (Takara Bio, Shiga, Japan) and sequenced with the M13 reverse primer from at least 12 clones.

The procedure for BSP was previously described by Clark et al. (1994) (link). Briefly, genomic DNA of porcine eye
cells was isolated using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) and
treated with the CpGenome Turbo Bisulfite Modification Kit (Millipore) according to
the manufacturer's instructions. Nested PCR using Taq Plus PCR Master Mix (TIANGEN,
Beijing, China) was performed for the amplification of the H19 DMR3
and IGF2 DMR1/2. The primer sequences are listed in Table 2. PCR products were purified and subjected
to BSP (10 positive clones) and Combined Bisulfite Restriction Analysis (COBRA),
which have been described in a previous study (Watanabe et al., 2010 (link); Huntriss et al., 2013 (link)).

Bisulfite conversion and COBRA were performed according to previous study (Chen et al. 2014 (link)). Briefly, for bisulfite modification, genomic DNA from different tissues and germ cells was treated with the CpGenome™ Turbo Bisulfite Modification Kit (Millipore) and EZ DNA Methylation-Direct TM Kit (Zymo Research), respectively. Primer sequences used for BSP are described in Additional file 1: Table S1. The PCR products were subjected to T vector cloning (positive clones, n = 10) and sequencing analysis, which showed heterozygosity at the (C/T) peak of Nnat. BSP products were also digested by a restriction enzyme BstUI (Thermo Scientific, MA, USA) for COBRA analysis. DNAMAN (LynnonBiosoft) and the online software tools MethOrimer and BiQ Analyzer were used for methylation analysis in this study.

Genomic DNA was extracted with the Genomic DNA Isolation kit (Foregene) and the bisulfite conversion reaction was performed using CpGenome Turbo Bisulfite Modification kit (Millipore), in accordance with the manufacturer′s instructions. PCR amplification of bisulfite-treated DNA was carried out with PCR Easy (Foregene). The amplified products were cloned and sequenced. The primers used in the PCR amplification are shown in S2 Table.