Extended Data Fig. 1. Differential chromatin accessibility and gene expression in young and old MuSCs.

a, Gating strategy for fluorescence-activated cell sorting (FACS) isolation of muscle stem cells (MuSCs). Purity of isolated MuSCs is > 98% as assessed by staining for Pax7 of cells fixed one hour after plating. (Scale bar, 50 μm) b-d, Representative confocal immunofluorescence images of MuSCs of young and old female mice (Scale bar, 5 μm). RFUs of H3K9me3 (b), H3K9me2 (c), or HP1α (d) were normalized to fluorescence intensities of total H3 (n=4). e, Pearson correlation of ATAC-seq profiles from young and old MuSCs (n=2). A total of about 60,000 signal summits were found in each sample. f, Representative heat maps of ATAC-seq tag intensity 3kb around transcription start sites (TSSs) in young and old MuSCs. g, Read density within 3 kb upstream of TSSs and 3 kb downstream of TESs. h, Hierarchical clustering of peak enrichment patterns between young and old MuSCs. i, PCA plot of RNAseq data from freshly isolated young and old MuSCs. j, Volcano plots of differentially expressed genes (DEGs) between young and old MuSCs. Dashed lines indicate fold-change (log₂FC>0.5) and p-value cut-offs (P_adj < 0.05). Total 1,641 DEGs were found. Among the DEGs, 866 genes were highly expressed in old MuSCs, and 775 genes were highly expressed in young MuSCs. k, Normalized DESeq read counts of methyltransferases for H3K9 (Suv39h1, Ehmt1, Ehmt2, Setdb1, and Setdb2). (n=4) l, Normalized DESeq read counts of demethylases for H3K9 (Kdm3a, Kdm3b, Kdm4a, Kdm4b, Kdm4c, and Kdm7b). (n=4) DESeq normalization was conducted by using median-of-ratio method as described in the Methods. Data are shown as median and quartiles (b-d) and as mean $\pm$ SD (k, l). P values were calculated by two-sided unpaired Student’s t-tests (b-d, k, l). *P < 0.05; **P < 0.01; ***P < 0.001. Statistical details are provided in Source Data.