Extended Data Fig. 3: Thymic epithelial cells are characterized by four subsets of mTEC and a single cTEC subset.

a, Heat map showing a metacell analysis of 2,341 thymic epithelial cells (CD45⁻EpCAM⁺), featuring the 73 most variable genes, from 15 biological replicates of 4–6-week-old mice. Colour bar represents separation of 36 metacells into five main populations. b, Two-dimensional graph representation of the metacell model in Fig. 1a (see Methods). Big circles represent metacells, and are colour-coded as shown in a. c, FACS index sorting measurement of LY51 and UEA1 in epithelial cells. Cells are coloured based on cluster association as determined in a. Dashed lines outline LY51⁺UEA1⁻ and LY51⁻UEA1⁺ gates. d, Fraction of TEC subsets out of LY51⁺UEA1⁻ and LY51⁻UEA1⁺ populations, assessed by gating single cells on index sorting protein measurements of UEA1 and LY51 in c. e, Controlling for batch effect as determined by the relative share of each batch in all metacells. Batches are ordered by biological replicate (marked by dashed lines) and sorting scheme (either CD45⁻ or CD45⁻EpCAM⁺). f, g, Single molecule FISH assay on 5–8-μm-thick cryosections (see Methods) using fluorescent probes against the genes Epcam and Sbsn (f) or Avil (g). Blue, DAPI. The experiments were repeated independently four times with similar results. h, Immunofluorescence images of the protein markers: PIGR and DCLK1. Medulla (M) and cortex (C) are separated by dashed lines, distinguished by nuclei density. Blue, DAPI. f–h, Scale bars, 20 μm. The experiments were repeated independently twice with similar results. i, Projection of representative differentially expressed genes onto the two-dimensional graph of epithelial cells.