Extended Data Fig. 8: C/EBPα single-cell RNA-seq supporting data.

a. Characterization of scRNA-seq clusters using the data for various stages of B cell macrophage differentiation from a previous study. Average expression for each cluster was normalized by vst and centered (z-score). K-means clustering was used to define the heatmap clusters. b. Quantification of the cluster’s genes for each k-cluster of the heatmap. Based on the quantification and expression profile of the heatmap the single-cell clusters were manually assigned. c. RNA velocity stream plot was embedded to pre-computed UMAP plot. The streamlines represent velocity vector field. The pseudotime plot (bottom right) illustrates the relative time relationship between the cells. d. Quantification of mEGFP-positive cells in the initial clusters. Cluster 0 and 2 contain virtually no mEGFP-positive cells, and were therefore removed from downstream analyses. e. Sample proportions for each cluster. Differentiating macrophage 1 is wild type-specific and Differentiating macrophage 2 is AroPERFECT IS15-specific. AroPERFECT IS10 cells are absent from the macrophage clusters. f. (left to right) Combined UMAP coloured CD14 and PTPRC, CD19 and ITGAM (MAC1) gene expression. These markers are associated with macrophage differentiation. g. Top 5 differentially expressed genes per cluster. These gene show specific expression signatures associated with each cluster and could be used as differentiation stage markers. h. Stacked violin plots for select DEG genes for Late macrophage cluster between AroPERFECT IS15 and wild type. Most genes seem to be expressed in other cluster with the exceptions of MMP9. CSF3R and CFD which seem to be macrophage and C/EBPα wild type specific while IL2RA is macrophage and C/EBPα AroPERFECT IS15 specific. i. Volcano plot of differentially expressed genes in the Late Macrophage cluster for wild type vs AroPERFECT IS15 samples. Differentially expressed target genes (Benjamini–Hochberg method, P < 0.05) are highlighted in blue. j. Flow cytometry analysis of GFP expression in RCH-rtTA clonal cell lines expressing GFP-tagged versions of C/EBPα. Data normalized to mode. k. Principal component analysis of the ChIP–Seq peak profiles for wild type and AroPERFECT IS15 C/EBPα-expressing cells 24 h and 48 h after induction of C/EBPα expression (PC1 vs. PC2). l, n. C/EBPα AroPERFECT IS15 shows enhanced binding at the CEACAM gene cluster (l) and at the FCGR2A locus (n). Displayed are genome browser tracks of ChIP–Seq data of C/EBPα wild type and AroPERFECT IS15 in RCH-rtTA cells, 24 and 48 hours after C/EBPα expression. Coordinates are hg38 genome assembly coordinates. m, p. Combined UMAP coloured on CEACAM8 and CEACAM1 (m) and FCGR2B and FCGR2A (p) expression. n, q. Flow cytometry analysis of CD66 (n) and FCGR2A (q) expression in differentiating GFP + RCH-rtTA cells 0 h and 48 h after induction of C/EBPα overexpression. Data normalized to mode.