Genomic DNA extraction and bisulfite conversion
Genomic DNA was extracted from 100 µL EDTA-2Na-treated blood using the DNeasy Blood & Tissue Kit (Qiagen Inc., Tokyo, Japan). Extraction was performed according to the manufacturer’s protocol. To adjust for the genomic DNA concentration, the elution volume was 100 µL for whole blood and 150 µL for buffy coat. The concentration of extracted DNA was measured using the NanoDrop 2000c spectrophotometer (Thermo Scientific, Tokyo, Japan). Extracted genomic DNA was stored at −30°C, then bisulfite converted using the EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions, and finally adjusted to 5 ng/µL.
Selection of target genomic locations
We selected target genomic locations adjacent to 12 genes, namelyGSE1 , VGF , SLC12A5 , SCGN , KCNK12 ,OTUD7A , BCL6B , POU4F2 , ELOVL2 , RALYL ,KISS1R , and CAPS2 (Table 1). The methylation levels of CpG sites adjacent to these genes change with age in humans (Day et al. 2013; Florath et al. 2014; Bekaert et al. 2015; Lowe et al. 2018), dogs (Ito et al. 2017; Yamazaki et al. 2021), and cats (Qi et al. 2021). We identified homologous sequences in the genomic regions of brown bears containing the target CpG sites using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information.