HSCs undergo global chromatin relaxation and H3K27 acetylation in response to replication stress. (A) Experimental schematic. HSCs (CD48⁻CD150⁺LSK) from WT C57BL/6J mice were sorted and cultured with DMSO, Gem, 0.5 µM, or HU, 100 µM for 48 h, followed by RNA-seq, ATAC-seq, and CUT&Tag-seq. (B) Principal component analysis of whole-transcriptome RNA-seq read counts in HSCs. (C) Representative volcano plot from RNA-seq analysis between the DMSO and Gem/HU-treated HSCs (n = 3 and 4 samples, respectively). All significantly changed genes (greater than twofold change, P < 0.05) are indicated by red dots. (D) GSEA from RNA-seq data was conducted (hallmark gene sets). The top 10 downregulated gene sets and the normalized enrichment score are presented here. (E) Representative heatmaps show ATAC-seq peaks ± 5 kb from transcriptional start site (TSS) in HSCs. Top, line plots of relative quantifications based on average intensities of all ATAC peaks detected. (F) The violin plots show the statistics and quantification of genomic-wide ATAC signal (log₂[normalized RPKM]) (n = 2 samples per group). (G) Representative heatmaps and line plots show H3K4m3 or H3K27ac CUT&Tag peaks ± 5 kb from TSS in HSCs. (H) The violin plots show the statistics and quantification of genomic-wide CUT&Tag signal in HSCs (n = 2 samples per group). (I) The violin plots show the statistics and quantification of ATAC and H3K27ac CUT&Tag signal at genomic regions (±5 kb from TSS) of IEGs and others in HSCs. The list IEGs is presented in Table S1. Others indicate a random gene set (56 genes). (J) Distribution of ATAC and H3K27ac CUT&Tag peaks across the indicated gene loci. The data presented in panels B–J are representative of two independent experiments. The results shown in panels F, H, and I are illustrated as violin plots. Statistical analyses were performed using a two-tailed unpaired Student’s t test. P values are presented as follows: *P < 0.05, ***P < 0.001; n.s., not significant. RPKM, reads per kilobase per million mapped reads.