DNA methylation level was analysis by MethylTarget™, an NGS-based multiple targeted CpG methylation analysis method. Specifically, the genomic regions of interest were analyzed and transformed to bisulfite-converted sequences by geneCpG software. PCR primer (Table 1 ) sets were designed with the Methylation Primer software from bisulfate converted DNA [7 (link)].
Genomic DNA (400 ng) was subjected to sodium bisulfite treatment using EZ DNA Methylation™-GOLD Kit (Zymo Research) according to manufacturer's protocols. Multiplex PCR was performed with optimized primer sets combination. A 20 μl PCR reaction mixture was prepared for each reaction and included 1x reaction buffer (Takara), 3 mM Mg2+, 0.2 mMdNTP, 0.1 μM of each primer, 1 U HotStarTaqpolymerase (Takara), and 2 μl templates DNA. The cycling program was 95°C for 2 min; 11 cycles of 94°C for 20 s, 63°C for 40 s with a decreasing temperature step of 0.5°C per cycle, and 72°C for 1 min and then followed by 24 cycles of 94°C for 20 s, 65°C for 30 s, and 72°C for 1 min; 72°C for 2 min [7 (link)].
Genomic DNA (400 ng) was subjected to sodium bisulfite treatment using EZ DNA Methylation™-GOLD Kit (Zymo Research) according to manufacturer's protocols. Multiplex PCR was performed with optimized primer sets combination. A 20 μl PCR reaction mixture was prepared for each reaction and included 1x reaction buffer (Takara), 3 mM Mg2+, 0.2 mMdNTP, 0.1 μM of each primer, 1 U HotStarTaqpolymerase (Takara), and 2 μl templates DNA. The cycling program was 95°C for 2 min; 11 cycles of 94°C for 20 s, 63°C for 40 s with a decreasing temperature step of 0.5°C per cycle, and 72°C for 1 min and then followed by 24 cycles of 94°C for 20 s, 65°C for 30 s, and 72°C for 1 min; 72°C for 2 min [7 (link)].
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