Bisulfite sequencing and quantitative RT–PCR analyses to validate alterations of DNA methylation and gene expression levels for six muscle-related genes, MYF6 (A), MUSK (B), MSTN (C), TRIM72 (D), CHRNA1 (E) and SGCA (F), during myogenic differentiation. Open and closed circles represent unmethylated and methylated CpG sites, respectively. Asterisks indicate the position of the CpG site that was found to be hypermethylated in MB_day15 compared with MB_day1 in the analysis of Infinium HumanMethylation450 BeadChip data [the corresponding name (IlmnID) of the probe is listed in Supplementary Material, Table S12]. Each row of circles corresponds to an individual clone sequenced. For each gene, the methylation ratios (%) of the CpG sites examined are shown in the table underneath. Gene expression levels in MB_day3, MB_day8 and MB_day15 relative to MB_day1 were displayed as bar charts. Each bar represents the mean fold change and the standard error of technical replicates (n = 3).