Single-cell transcriptomics reveals a temporal association between FcεRI and the mast cell gene signature. (A) Representation of the cells in the first data set relative to the cells in the prospective second data set. (B) Experimental approach to hashtag a cell subset with a PE-oligonucleotide barcode. (C) Post-sort flow cytometry analysis of pooled cells, constituting Lin⁻ CD34⁺ c-Kit⁺ cells without and Lin⁻ CD34⁺ c-Kit⁺ FcεRI⁺ cells with the PE-oligonucleotide hashtag. (D) UMAP visualization of the entire Lin⁻ CD34⁺ c-Kit⁺ data set (4063 cells). The colors indicate Leiden clusters. Panels E and G and supplemental Figure 3 provide landscape annotation. Population A (Pop A) consisted of 2 main clusters, Pop A1 and Pop A2. (E) Gene expression levels of mast cell genes and FcεRI subunit genes. (F) Histogram showing the distribution of the PE-oligonucleotide in the single-cell transcriptomics data, resembling the flow cytometry data in panel C. Gray bins represent ambiguous events. (G) Plots showing the PE⁻ Lin⁻ CD34⁺ c-Kit⁺ cells and PE⁺ Lin⁻ CD34⁺ c-Kit⁺ FcεRI⁺ cells (population A). (H) Visualization of highly dynamic genes along the mast cell differentiation trajectory. Cells are ordered according to cluster and pseudotime. (I) Visualization of dynamic genes that positively correlate with rank ordering, also referred to as Pop A signature genes. (J) Plot showing the temporal upregulation of mast cell genes and FcεRI subunit genes. The blue to yellow color gradients in panels H and I specify low to high gene expression. One buffy coat, independent of the buffy coat used for Figure 1, was used to produce the data presented in panels C to J. Ab, antibody; Ery, erythroid; Ly/Mono/DC, lymphocyte/monocyte/dendritic cell; MNCs, mononuclear cells; MPP, multipotent progenitor; Neu, neutrophil; scRNA-seq, single-cell RNA-sequencing.