Epityper system

The DNA methylation markers selected for this study were seven CpG sites from the genomic regions: ELOVL2, ASPA, PDE4C, FHL2, CCDC102B, MIR29B2CHG and chr16:85395429 (GRCh38), included in a previous age prediction model initially created for adult samples [26] . The Agena Bioscience EpiTYPER® system (San Diego, CA, USA) is a bisulfite-treatment-based method for detection and quantification of DNA methylation using MassARRAY® mass spectrometry [46] . The bisulfite conversion step is performed with the EZ DNA Methyl-ation™ Kit (Zymo Research), with input of 300 ng of genomic DNA, following manufacturer's guidelines, to produce 40 µL of converted DNA. That means that from the samples normalized to 10 ng/µL, 30 µL were used for bisulfite conversion. From the final 40 µL of converted DNA, 1 µL was used for subsequent EpiTYPER analyses. EpiTYPER DNA methylation data for the present study were obtained from two previous publications [26, 42] . EpiTYPER detects methylation levels as CpG sets, comprising one or multiple CpG positions in the same cleavage fragment. Therefore, multiple CpGs in a set will be detected when they are closely positioned on the targeted fragment. Herein, we use the term CpG site whether one CpG, or a cluster of CpGs is detected in a single short DNA fragment.