Polymerase chain reaction and pyrosequencing
Polymerase chain reaction (PCR) and subsequent pyrosequencing were conducted following previously reported methods (Yamazaki et al. 2021). PCR was performed using the TaKaRa EpiTaq HS (for bisulfite-treated DNA) (Takara Bio Inc., Shiga, Japan). PCR and pyrosequencing primers, listed in Supplementary Table S_M2, were designed using Methyl Primer Express v1.0 (Thermo Fisher Scientific, San Jose, CA, USA) and PyroMark Assay Design v2.0.2.5 (Qiagen Inc.). PCR was conducted in two steps so that biotin-modified primers could be used in any region (Yamazaki et al. 2021).
The first PCR step was performed in a total volume of 15 µL containing 0.75 µL of the genomic DNA sample (diluted to contain 3.75 ng DNA), 0.075 µL TaKaRa EpiTaq HS, 1.5 µL 10× EpiTaq PCR Buffer (Mg2+ free), 1.5 µL MgCl2, 1.8 µL deoxynucleoside triphosphate (dNTP) mixture, 0.3 µL each of the forward and reverse primers (10 µmol/L), and 8.775 µL molecular-grade water (Nippon Gene, Tokyo, Japan). The PCR conditions were 98°C for 1 min, followed by 35 cycles of 98°C for 10 s, annealing temperature (listed in Supplementary Table S_M2) for 30 s, and 72°C for 30 s. Each sample was run in duplicate. The second PCR step was performed in a total volume of 15.13 µL containing 0.1 µL of the first PCR product, 0.075 µL TaKaRa EpiTaq HS, 1.5 µL 10× EpiTaq PCR Buffer (Mg2+ free), 1.5 µL MgCl2, 1.8 µL dNTP mixture, 0.3 µL of the forward primer (10 µmol/L), 0.06 µL of the reverse primer (10 µmol/L), 0.27 µL of the biotin-modified primers (10 µmol/L), and 9.525 µL molecular-grade water (Nippon Gene). The PCR conditions were 98°C for 1 min, followed by 35 cycles of 98°C for 10 s, annealing temperature (listed in Supplementary Table S_M2) for 30 s, and 72°C for 30 s. The success of the first and second PCR amplifications was confirmed by electrophoresis on a 2% agarose gel.
To determine the methylation levels of the target CpGs, pyrosequencing was performed using PyroMark Q48 software (Qiagen Inc., Tokyo, Japan) with PyroMark Q48 Advanced Reagents (Qiagen Inc.) according to the manufacturer’s instructions. As PCR for each sample was conducted in duplicate, the average value was taken as the methylation level for that sample. Initially, as a screening step, each target CpG site was analyzed across nine samples to identify age-related genomic locations, i.e., those showing a correlation between age and DNA methylation levels. These samples were selected from captive bears, with the numbers of individuals balanced according to age and sex. From 12 candidate genomic locations, locations with R2 values greater than 0.8 and a range of methylation level changes greater than 20% were selected for further analysis. Finally, we analyzed the remaining blood samples based on the selected genomic locations and calculated Pearson’s product-moment correlation coefficients and p values.