Fig 2: Stem/progenitor cell populations in EphA2 knockout mice compared to wild type littermates.(A) Gating for Progenitors (lineagelowcKithighSca-1-), LKS+ cells (lineagelowcKithighSca-1+ enriched for hematopoietic stem cells), CMP (lineagelowcKithighSca-1-CD34+FCGRII/IIIlow), MEP (lineagelowcKithighSca-1-CD34-FCGRII/III-) and GMP (lineagelowcKithighSca-1-CD34+FCGRII/IIIhigh) presented as percentage of Lineagelow cells. CD34/CD135 gating for MPP (LKS+CD34+CD135+), ST-HSC (LKS+CD34+CD135-) and LT-HSC (LKS+CD34-CD135-). CD48/CD150 gating for LT-HSC (LKS+CD150+CD48-) and MPP (LKS+CD150-CD48+). (B) There were no significant differences in progenitors, LKS+, CMP, MEP or GMP population in the EphA2 knockout mice compared to wild type control mice (n = 15, 3 independent experiments). (C) There were no significant differences in LT-HSC and MPP population gated by CD34/CD135 antigens in the EphA2 knockout mice compared to wild type control. There were significantly more (P = 0.0012) ST-HSCs in EphA2 knockout bone marrow compared to wild type control (n = 15, 3 independent experiments). (D) There were no significant differences in LT-HSC and MPP population gated using CD48/CD150 antigens in EphA2 knockout mice compared to wild type control (n = 15, 3 independent experiments). Each dot corresponds to one individual mouse. The data represent mean ± SEM. Unpaired t test was performed for statistical analyses.

Fig 3: Single-cell DNA methylation profiles deliver a high-resolution map of mouse hematopoiesis.A. uMAP of DNA methylation data for HSPCs from the LARRY main experiment, the LARRY replicate experiment and from native hematopoiesis. Batch correction was applied prior to uMAP. Colors highlight groups identified from unsupervised clustering. B. uMAP of transcriptomic data from the same experiments. C. uMAP as in A, highlighting relative methylation state of cells across all CpGs that are methylated in HSCs or MPP3/4 in bulk data. D. uMAP as in A, highlighting relative methylation states of CpGs located in vicinity of TFBS. E. Surface protein expression of Sca1, c-Kit, CD135, CD201, CD48, and CD150. The CD135/CD201 and CD48/CD150 plots only show Lin−Sca-1+c-KIT+ cells and the color codes corresponds to the cell type clusters defined in the DNAm uMAP F. Enrichment of CpGs specifically unmethylated in a cell-type cluster according to the vicinity to the annotated TFBS.

Fig 4: DNA methylation jointly encodes cellular differentiation and clonal identity.a, Schematic of experiments M.1 (LARRY main experiment) and M.2 (replicate LARRY experiment). b, Overview of the 453 CpGs covered by our scTAM-seq panel in mice. Variably methylated CpGs were selected from bulk whole-genome bisulfite sequencing data22. DMC, differentially methylated CpG; IMC, intermediately methylated CpG; WSH, within-sample heterogeneity (see Extended Data Fig. 1c for definition). c, UMAP of DNA methylation data for HSPCs from experiments M.1–M.3. Batch correction was applied before UMAP. Colours highlight groups identified from unsupervised clustering. Annotations are based on d–f. d, DNA methylation UMAP as in c, highlighting the average, relative methylation state of cells across all CpGs that are methylated in HSCs or MPP3/MPP4 cells in bulk-sequencing data22. e, Enrichment analysis of TFBSs near CpGs specifically unmethylated in a cell-type cluster. See the section ‘Data integration and annotation of cell states’ in the Methods. f, Normalized surface-protein expression of SCA1, KIT, CD135, CD201, CD48 and CD150. The CD135–CD201 and CD48–CD150 plots only show LSK cells. Colour indicates cell states per c. g, UMAP of DNA methylation data from HSPCs from experiment M.1. Colour indicates cell states per c. h, Same UMAP as in g, highlighting clones as defined from LARRY barcodes. LARRY barcodes were read out from DNA as part of scTAM-seq. i, Scatter plot depicting, for n = 453 CpGs, the average methylation rate, the statistical association with surface-protein expression and the statistical association with the LARRY clonal barcode (P value from a two-sided chi-squared test). The CpGs in the upper and lower central rectangle were defined as static or dynamic CpGs, respectively. j, Bar chart depicting the percentage of static and dynamic CpGs annotated as enhancer or heterochromatin. DMC, differentially methylated cytosine; IMC, intermediately methylated cytosine; MEP, megakaryocyte–erythroid progenitor cells; WSH, within-sample heterogeneity. The scTAM-seq schematic in a was adapted from ref. 5 under a Creative Commons licence CC BY 4.0.Source data