Massarray epityper software

Genomic DNA was extracted from 12 lungs of control and ALI model mice by using DNeasy Blood and Tissue kit (QIAGEN, Germany, 69504) according to manufacture’s instructions. The quality and quantified were evaluated by gel electrophoresis and a NanoDrop spectrophotometer (Thermo, USA). The genomic DNA from each sample was treated with sodium bisulfite using an EZ DNA methylation kit (Zymo Reasearch, USA). The MassARRAY platform (The Beijing Genomics Institute, China) was used for quantitative analysis of MAPK4 methylation. We used the primers (5′-aggaagagagGGGTGGGTTTTATTAGAGATAGTGG-3′, 5′-cagtaatacgactcactatag-ggagaaggctAATCTAAATCCCAACTAAATAATCCC-3′) to amplify the region of each promoter. Altogether, 35 CpG sites were tested in this region. The spectra methylation ratios of each CpG site were generated by MassARRAY EpiTYPER software (Agena, USA).

We randomly recruited 118 hypertension cases and 149 health controls from the community to validate the CpGs mapped to COL5A1 and WNT3A in EWAS. The cases were defined as those with SBP ≥ 140 mmHg and DBP ≥ 90 mmHg. The subjects with a history of diabetes, obesity, cancer, stroke, and cardiovascular disease were excluded. The participants were interviewed when blood samples were taken and stored under − 80 °C for DNA methylation analysis. We designed the primers for COL5A1 and WNT3A gene to cover the region with the most CpGs (p < 0.05) in EWAS. The mass spectra of cleavage products were collected using the MALDI-TOF mass spectrometry based on the MassARRAY System (Bio Miao Biological Technology, Beijing, China), and the spectra’s methylation ratio was generated by MassARRAY EpiTYPER software (Agena Bioscience, San Diego, California). The DNAm of CpGs between the two independent groups was compared by Wilcoxon rank-sum test. Binary logistic regression model was applied to evaluate the association of each CpG with hypertension while adjusting for BMI, triglyceride (TG), and fasting blood glucose (FBG). The p < 0.05 was set as statistically significant.

To validate CpGs located at FBLN1 and ABHD14B, we recruited 117 participants with low HGS (defined as male<28 kg, female<18 kg) and 117 healthy controls from the community. The method for HGS testing is consistent with the aforementioned approach. Participants attended interviews and venous blood samples were collected for DNAm level measurements. Primers for the FBLN1 and ABHD14B genes were designed to cover the majority of the CpGs identified in EWAS. The cleavage products were analyzed using MALDI-TOF mass spectrometry based on MassARRAY System (Bio Miao Biological Technology, Beijing, China). Subsequently, the resulting spectra were processed using the MassARRAY EpiTYPER software (Agena Bioscience, San Diego, California) to determine the methylation ratio. The Wilcoxon rank-sum test was used to compare the DNAm levels of GpGs between the two groups. The association between each CpG and low HGS was evaluated by logistic regression, with adjusting for age, sex, and BMI. The significance level was defined as p < 0.05.