Fig. 4. Transcriptome profiling of Setd5-deficient HSCs reveals altered stem cell property and cell cycle signature.

A Heat map showing differential expression of 443 genes in Setd5^fl/fl and Setd5^CKO SLAM-HSCs, |Log₂foldchange | > 1, p value < 0.05; n = 3. B GSEA analyses for genes affected in the SLAM-HSCs of Setd5^fl/fl and Setd5^CKO mice. NES, normalized enrichment score. C Relative expression levels of cell cycle and multi-potency genes in HSC cells; n = 3. mRNA levels were normalized to the expression of 18 s. D. Experimental design for single cell sequencing with Smart-seq2, LSK⁺ cells were indexed sorted into 96-well plates containing lysis buffer. E LSK⁺ cells were projected onto Nestorowa et al. data [30]. F UMAP visualizations of single cell transcriptomes of identified clusters using Seurat. G–I Diffusion maps of all cells were colored according to the expression levels of selected genes. J, K Histograms showing the compositions of surface markers defined four populations and transcriptome-defined five clusters by ten cell types annotated with Nestorowa et al. data. C1-C5 means cluster 1-5. Ten annotated cells including E-SALM (EPCR⁺CD48^–CD150⁺), LT-HSC (Lin^–Sca1⁺c^-Kit⁺CD34^–Flt3^low), ST-HSC (Lin^–Sca1⁺c-Kit⁺CD34⁺Flt3^lowCD48^–CD150^–), MPP1 (Lin^–Sca1⁺c-Kit⁺CD34⁺Flt3^lowCD48^–CD150⁺), MPP2 (Lin^–Sca1⁺c-Kit⁺CD34⁺Flt3^lowCD48⁺CD150⁺), MPP3 (Lin^–Sca1⁺c-Kit⁺CD34⁺Flt3^lowCD48⁺CD150^–), LMPP (Lin^–Sca1⁺c-Kit⁺CD34⁺Flt3⁺), CMP, GMP and MEP. L GSEA of LT-HSC, proliferation and quiescence signatures comparing between Setd5^CKO and Setd5^fl/fl in HSC enriched cluster 1 and cluster 2. M, N Proportion of SLAM-HSCs, HPC1s and five clusters identified with Seurat in each of the cell cycle stages between two groups.