Fig. 2: RNA velocity analysis links Ly6D⁺Siglec-H⁺ precursors and pre-cDCs.

Single-cell RNA sequencing was performed for CD115⁺ CDP, CLP, Ly6D⁺ Siglec-H^– LP, Ly6D⁺Siglec-H⁺ LP, Siglec-H^– pre-cDC, lo-lo, lo-hi precursors and pDCs sorted from Lineage-depleted BM cells. a Diffusion map of 675 DC precursors and related cells with cells highlighted by their identity according to cell sorting. b Ly6d gene expression overlayed onto the diffusion map. c Comparison of Zbtb46-eGFP expression in pre-cDC, lo-lo, lo-hi precursors, and pDCs in BM cells of heterozygous Zbtb46^gfp mice measured by flow cytometry. Histograms show the Zbtb46-eGFP fluorescence signal (normalized to mode), WT cells were used as a negative control. Representative results of 3 independent experiments. d RNA velocities projected onto the diffusion map as streamlines with scvelo. Louvain clusters are indicated by colors and numbers. Clusters were annotated according to their gene expression and sorted cell type composition. e Partition-based graph abstraction (PAGA) with velocity directed edges computed with scvelo. Solid black arrows indicate probable velocity-inferred transitions of high confidence. Dotted lines indicate clusters that are connected by transcriptome similarity, but do not have sufficient support by RNA velocity to indicate confident transitions. f Expression heatmap of manually selected marker genes, scaled between 0 and 1 in Louvain clusters 0 to 6. g From left to right: Spliced/unspliced phase portraits, RNA velocity, and expression level of the indicated genes overlaid onto the diffusion map.