Figure S3.

Role of Ebf1 deficiency in myeloid-biased transcriptome. Related to Figs. 4 and 5. (A) PCA of bulk RNA-seq data from MPP3 and MPP4 cells of Ebf1^WT and Ebf1^KO conditions; based on DE genes. n = 4 biological replicates. (B) MA plots of MPP3 and MPP4 cells comparing Ebf1^WT and Ebf1^KO transcriptomes. Dots in blue represent genes with an FDR <0.1. The y axis represents shrunken log₂ fold change (FC), capped at |log₂ FC| 2, and the x axis represents log₁₀ basemean. (C and D) Comparison of chromatin accessibility changes to gene expression changes in MPP3 (C) and MPP4 (D) cells upon Ebf1 deletion. The y axis represents shrunken log₂ fold changes of gene expression and the x axis represents log₂ fold changes of peak read counts in Ebf1^WT versus Ebf1^KO MPP4 cells. Left: Dots represent DA peaks in MPP3 (C) and MPP4 (D) cells. Right: Dots represent DE genes and ATAC peaks that overlap their TSS (TSS peaks). (E) CFU assay performed with 150 sorted MPP3 and MPP4 cells plated in triplicate in methocult. Total number of colonies formed after 10–12 d of plating. Biological replicates Ebf1^WT and Ebf1^KO n = 6. Statistical significance was determined by unpaired t test. (F) Heatmap showing gene expression of DE genes in MPP3 and MPP4 cells, between Ebf1^WT and Ebf1^KO conditions. Genes are organised into MPP3-specific, common, and MPP4-specific DE genes. Genes with an annotated C/EBPα ChIP peak (original data from Hasemann et al. [2014]; Pundhir et al. [2018]) are labeled in blue. Heatmap scale represents z-scores calculated separately for MPP3 and MPP4 cells. (G) qPCR analysis of Cebpa expression in Ebf1^WT and Ebf1^KO MPP3 cells transfected with a control or a Cebpa siRNA pool. Cebpa mRNA expression relative to Actb was normalized to the control transfected samples. Data are represented as mean ± SEM. Ebf1^WT and Ebf1^KO n = 4. (E and G) Data are from >2 independent experiments.