Figure 1.

Single-cell RNA sequencing of FDCs: clusters and validation. (a) Schematic of the scRNaseq workflow. Tonsils were gently digested to a single-cell suspension, enriched for FDCs by Percoll gradient centrifugation, sorted in 384-well CEL-seq2 plates, and aligned and analyzed in python and R. (b) Gating strategy. SSC-A^high cells were selected, then live CD45⁻CD31⁻ cells. From here, FDCs were defined as PDPN⁺CD35⁺. (c) Unbiased Uniform Manifold Approximation and Projection (UMAP) clustering was used to determine similar cell types; doublets, B cells, and keratinocytes were removed. (d) Heatmap of most differentiating genes in scRNaseq with the same genes in the microarray. Yellow is high expression; purple is low expression. (e) Known genes for the cell populations of interest. Complement receptors and CXCL13 set the FDCs apart, while TNFSF11 (RANKL) and IL-33 define MRC. FRCs were PDPN⁺, LTBR⁺, and PDGFRA⁺, as expected. Sctransform-normalized expression. (f) Cell cycle phases of the clusters as determined by G0-, G1-, and S-phase genes. Related to Fig. S1 and Fig. S2. scRNaseq data, n = 4; microarray data, n = 3 (biological replicates). BEC, blood endothelial cell; FSC-A, forward scatter area; LEC, lymph endothelial cell; SSC-A, side scatter area.