Extended Data Fig. 4: FACS and Fluidigm-based analysis of steady-state terminal granulopoiesis.

a, FACS plots demonstrating the gating strategy used for sorting. The populations sorted for scRNA-seq are indicated in shades of grey with accompanying cytospins. b, Violin plots of the gene and read-level metrics for each of the indicated libraries. Dashed lines indicate mean, lower and upper quartiles. Sample size (n = number of cells) is displayed at the top. c, Heat map of gene expression defined by ICGS (Fluidigm C1, excluding cell-cycle genes) in scRNA-seq data (n = 516 cells). Each column represents a single cell and each row represents a single gene. ICGS clusters are annotated (top). NK, natural killer T-cell progenitor. d, Joint UMAP plot of scRNA-seq data from c, separated as previously described¹⁹ and new Fluidigm captures show no detectable batch effects. e, Bar chart of the heat map in c displaying the incidence and amplitude of selected genes. f, Heat map of correlation between gene expression and each displayed cluster as generated by MarkerFinder (AltAnalyze software). g, FACS plots comparing expression of LY6G with the indicated surface marker. FMO, fluorescence minus one control. h, Heat map of cell cycle gene expression in ICGS-defined clusters in scRNA-seq data (n = 509 cells). Each column represents a single cell and each row represents a single gene. Gene expression clusters were generated in AltAnalyze and the ICGS clusters are annotated (top). FACS gates are annotated (bottom). LK CD34⁺, Lin^neg KIT⁺ SCA-1^neg CD34⁺. Key genes are indicated (right). i, Scatter plot representation of scRNA-seq data from h comparing the gene expression of G1-to-S phase transition genes with G2-to-M phase transition genes in each cell. Each point represents a single cell. j, Bar chart of the heat map in h, displaying the incidence and amplitude of selected genes. k, Heat map of correlation between gene expression and each displayed cluster as generated by MarkerFinder. l, Heat map of enrichment for Gene Ontology biological processes enriched in the granulocytic clusters from the Fluidigm scRNA-seq data from c with key processes indicated (right). m, Scatter plot representation of scRNA-seq data from c, in which each point represents a single cell. Reads per cell indicate RNA-seq by expectation–maximization (RSEM) transcript-aligned read counts for each cell library. Genes expressed per cell indicate the number of genes with a transcripts per million (TPM) >1 for each single-cell library. Data in a and g are representative of three biological replicates; data in f and k display Pearson correlation values.

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