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. 2022 Sep 3;13:5187. doi: 10.1038/s41467-022-32970-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a–f UMAP plots for scATAC experiments from old vs. young HSCs (a–c) and Clca3a1^high vs. Clca3a1^low HSCs (d, e). Motif accessibility of SPI1 and TEAD4 is color-coded. The pseudotime trajectory is included in c, f, respectively. g Scatter plot that shows the most variable motifs in the given comparisons. h, i Scatter plot showing the SPI1 and TEAD4 motif accessibility (as Z-score) of individual cells within the pseudotime trajectory for the indicated comparisons. j Expression of Spi1 in the different clusters of the CITE-Seq dataset. k Immunoblot for PU.1 in BM-HPC#5 after infection with the indicated shRNAs. Gapdh serves as loading control (experiment repeated once with similar results). The experiment was independently repeated three times. l Volcano plot for gene expression changes after PU.1 depletion (shPU1#1 and shPU1#2) in BM-HPC#5 cells. DEGs (padj < 0.05, log₂FC > 1 or log2FC < (−1), respectively) are colored in blue and red. padj = adjusted p-value. m Violin plots for the shPU1.up gene set activity in the indicated clusters from the scRNA-Seq dataset of young and old LSK cells (two-sided Wilcox test). n Violin plots for the shPU1.up gene set (n = 899 genes) and the shPU1.down gene set (n = 667 genes) comparing the expression in CLCA3A1^high vs. CLCA3A1^low LT-HSCs (two-sided Wilcox test). o Log₂ RPKM values of the indicated genes coding for HSC surface markers in CLCA3A1^high and CLCA3A1^low LT-HSCs, respectively. Data from RNA-Seq comparing CLCA3A1^high vs. CLCA3A1^low LT-HSCs (n = 3 per group, LSK CD34^neg CD135^neg). p Representative flow cytometry plots from young and old LSK cells, respectively. In the right panel (LSK old gated on CLCA3A1^low), the oLSK cells were additionally gated on low CLCA3A1 staining intensity. q Barplots summarizing the analysis outlined in p (n = 33 for young, n = 30 for old mice, one-way ANOVA with Tukey HSD post hoc test). Boxplots in m, n: bottom/top of box: 25th/75th percentile; upper whisker: min(max(x), Q_3 + 1.5 * IQR), lower whisker: max(min(x), Q_1−1.5 * IQR), center: median. Source data are provided as a Source data file.