DNA Extraction, Bisulfite Treatment, Methylation Array Methods
A commercially available kit (TIANGEN Biotech, Beijing, China) was used to extract genomic DNA from whole blood according to previous publications [22]. Genesky Biotechnologies Inc. performed bisulfite processing, methylation library construction, high-throughput sequencing and quality control [23]. CpG islands located between 2K upstream of the gene transcription start site and 1K downstream of the first exon were selected to measure methylation level. 18 CpG islands from the 9 screened aging-related genes were selected (2 from AREG, 2 from ATG3, 1 from E2F1, 3 from FOXO3, 1 from HDAC1, 3 from MMP2, 1 from NUF2, 3 from TGFB1 and 2 from TP53) according to our previous publications [18]. Then, bisulfite modification of DNA sample, methylation library construction and MethTarget were performed [18]. 856 CpG sites from 9 distinguishingly expressed aging-related genes in the methylation assay were detected. We only selected the original data with a sequencing quality value of Q>40 (basic sequencing error rate <0.1%), and the methylation percentage of each CpG site was presented.