Skewing of HSC AS genes reflects transcriptional activation in aged HSCs. (A) Skewing of HSC AS genes. Volcano plot depicting expression levels of AS genes; 181 upregulated and 39 downregulated genes are represented. Dots represent the average of either adjusted P values (y-axis) and FCs (x-axis) per gene in different studies. (B) Protein levels of RNA polymerase II in individual young and aged LT-HSCs. Right panel shows the mean fluorescence intensity (MFI) quantification of young (n = 120 cells) and aged HSCs (n = 139 cells). Scale bars, 5 μm; mean ± standard deviation is shown. ****P < .0001. (C) Aged HSCs demonstrate more age-specific chromatin accessible sites than young HSCs. Volcano plot depicting differentially accessible sites (peaks) between young and aged HSCs measured by ATAC-seq. Dot colors demonstrate peaks either in genomic regions overlapping with coding (green) or noncoding (purple) annotations. Peaks with negative FC values are significantly more accessible in young HSCs, whereas peaks with positive FC values are more significantly accessible in aged HSCs. Gene symbols annotate peaks which overlap with gene bodies from some AS genes. (D) Transcriptional program is associated with ATAC-seq of young and aged HSCs. GO analysis of functional process for overlapping aged-specific ATAC-seq accessible sites with genes from the aging list. Horizontal bar represent individual GO terms (y-axis) and their FDR values (x-axis). Numbers in each bar demonstrate the number of genes found per GO term. GO terms are selected based on a FDR <0.05 cutoff.