Epitect bisulfite kit protocol

Genomic DNA extraction was performed using the TaKaRa Genomic DNA Extraction Kit (TaKaRa Co., Dalian, China). Genomic DNA (2 ug per sample) was bisulfite modified with the Epitect Bisulfite Kit Protocol (Qiagen), and the modified DNA was amplified using the following primers: BSQ1 forward, 5′-gaaggatttcggttaatttgggg-3′, and reverse, 5′-caaactcgccaaataactacctacg-3′; and BSQ3 forward, 5′-ggttgattatttgaggttaggtgtt-3′, and reverse, 5′-aaaacaattttcaaccaaccatc-3′. The modified DNA was amplified by PCR using 0.2 µM of each primer, 2 units of Hot Start Taq DNA polymerase, and 0.2 mM of each dNTP per reaction. Cycling programs were 95°C for 10 minutes, and then 40 cycles of 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 30 seconds, followed by a 5-minute incubation at 72°C. The PCR products were examined by gel electrophoresis in 1.5% agarose to confirm that a single band had been obtained and were then sequenced by Invitrogen. Methylation-specific PCR (MS-PCR) was carried out on bisulfate-treated DNA. The primers used were Un-methylated KLF4 forward, 5′-ggttgattatttgaggttaggtgttt-3′, and reverse, 5′-cccaaataacaaaaattacaaacat-3′; and Methylated KLF4 forward, 5′- gttgattatttgaggttaggtgttc-3′, and reverse, 5′-cgaataacgaaaattacaaacgta-3′. Umbilical cord blood DNA was used as a negative control, and it was methylated in vitro by using the Sss1 (CpG) methylase (New England Biolabs).

Bisulfite sequencing was conducted as described [50 (link)]. Briefly, extracted DNA samples were bisulfite converted using the Epitect Bisulfite Kit Protocol (Qiagen, Düsseldorf, Germany). The sequencing service was provided by Tech Dragon Ltd., Hong Kong. The PCR primers were listed as follows: NKX6−2 forward: GGTTTGGAAAAGTTATTTG; NKX6−2 reverse: AAACTACACCTCAATAAAACC.