Zymotaq premix

Sequencing primers of NLRC4 were designed by using PyroMark Assay Design 2.0 software. The sequences were 5’-TGGGTAAGGTTATATAGGGTAAATAGA-3’ for the forward primer, 5’- Biotin-ATACAAAAAAATTTCTTCAATCCTCAACT-3’ for the reverse primer, and 5’-TATATAGGGTAAATAGATTATTAGT-3’ for the sequencing primer. All primers were synthesized by Sangon Biotechnology (Sangon, Shanghai, China). 200–500 ng of genomic DNA were bisulfite treated using the EZ DNA Methylation Gold ™ kit (Zymo Research, CA, USA) following the operation manual. Genes were amplified using Zymo Taq Premix (Zymo Research, CA, USA) in a total volume of 20 µL, incluing 10 µl Zymo Taq Premix, 6.5 µl DNase/ RNase-free water, 1 µl primer pairs (20 pM), and 2.5 µL bisulfite-converted DNA. Then sequencing was performed by loading PCR product (10 µl, about 250 ng template) and 3 µl magnetic beads into PyroMark Q48 Autoprep. Finally, the methylation status of each CpG site was analyzed using the Pyro Q CpG software.

Genomic DNA was extracted with a TIANamp Genomic DNA Kit (Tiangen, Beijing, China) and prepared with sodium bisulfate using an EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s protocol. Three separate bisulfite modification treatments were performed for each DNA sample. The BSP primers were designed by the online MethPrimer software [38 (link)] and are shown in Table 1. The PCR amplifications were performed in 20 μL volumes containing 50 ng of genomic DNA, 0.5 μM each of MSPF/MSPR primer (Table 1), and ZymoTaq Premix (ZymoTaq Premix, Zymo Research, USA) 10 μL. The PCR was performed using a program of 5 min at 95 °C, 38 cycles of 94 °C 30 s, annealing for 40 s at 50.4 °C and extension for 30 s at 72 °C. All PCR products were subcloned into the pMD19-T vector (Takara). Different positive clones for each subject were randomly selected for sequencing (Sangon, Shanghai, China). Finally, the sequences were analyzed using online QUMA software [39 (link)].

DNA treatment with sodium bisulfite was performed using the EZ DNA Methylation Kit (Zymo Research, USA) in accordance with the manufacturer’s protocol. Following modification, the DNA samples were diluted in 10 mL of distilled water and immediately used for bisulfite sequencing PCR (BSP). BSP primers were designed with Methyl Primer Express v1.0, and the sequences of the PCR primers utilized for amplifying the targeted products are presented in Table 1. Hot start DNA polymerase (Zymo Taq TM Premix, Zymo Research, USA) was utilized for BSP performed in 50 mL of reaction volume containing 200 ng/50 mL genomic DNA, 0.3–1 mM each primer, and 25 mL of Zymo Taq TM Premix. PCR amplification was carried out using a DNA Engine Thermal Cycler (Bio-Rad, USA) with the following program: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 57.5°C, and extension for 30 s at 72°C, with a final extension at 72°C for 10 min. The PCR products were subsequently gel purified using the Gel Purification Kit (Sangon, Shanghai, China), and the purified fragments were subcloned into the pMD-19T Vector (TaKaRa, China). Ten positive clones for each subject were randomly selected for sequencing (Sangon, Shanghai, China), and the final sequence results were processed using the BIQ-Analyzer.