Sort strategy, 10x Genomics workflow and identification of B cell subsets represented by UMAP clusters in 10x HCD1. (A) Gating strategy to sort live CD19⁺ cells. (B) TotalSeq antibodies and clones used for surface labeling of CD19⁺ B cells. (C) Demographic details of HCDs, cells captured, and sequencing depth. (D) UMAP plot demonstrating clusters generated from a PCA run on 2,000 differentially expressed genes from 10x HCD1. (E) Feature plots demonstrating lineage defining ADT (CITE-seq antibody) and transcript signal overlay on the UMAP plot. (F) Table of median IgM (IGM) expression within clusters representing naive cells (CD27⁻IgD⁺CD38^int). The top 30% of clusters were designated as IgM^hi and designated H in the column labeled ID. The remainder of clusters that were not IgM^hi are designated N. (G) Merged and pseudocolored clusters representing B cell subsets defined by ADT and gene signal of lineage defining targets. CSM, class-switched memory. (H) A 3D UMAP plot demonstrating merged and pseudocolored clusters representing B cell subsets from 10x HCD2. (I) A 3D UMAP plot demonstrating merged and pseudocolored clusters representing B cell subsets from 10x HCD3. FSC-A, forward scatter area; SSC-A, side scatter area.