scRNA-seq recapitulates aberrant differentiation of early BCPs in patients with IL-7Rα deficiency. (A) Preparation of the scRNA-seq data set. Mononuclear cells of BM samples of 2 healthy pediatric donors and 2 patients with IL-7Rα deficiency were isolated via Ficoll density centrifugation, depleted of dead cells, stained with CITE-seq antibodies, and enriched for BCPs using magnetic-activated cell sorting before preparing the single-cell library according to the 10x Chromium protocol, followed by sequencing using Illumina technology. (B) UMAP plot showing the distribution of the individual clusters of the entire data set of patient and control samples. (C) Individual UMAP plots showing the clusters and their distribution in the adjacent panel for each control and patient. Color coding is identical to that in panel B. (D) Violin plots showing the expression of the B-cell markers used for the annotation of the individual B-cell clusters. Bottom 3 panels represent CITE-seq markers. (E-F) Pseudotime analysis for the early (E) and late (F) BCP subsets. Left panels in panels C,E-F show pseudotime calculated as described in “Methods” and plotted on the UMAP of the respective data set; right panels show comparison of pseudotime values for controls vs patients. Black lines in the left plots in panels E-F denote the trajectories.