Clone 9c4

For fetal and postnatal tissues, single cells from digested tissue were resuspended in MACS buffer containing 10 μM of the ROCK inhibitor Y-27632 (Tocris). Human CD45 MicroBeads (Miltenyi) were used to deplete immune cells according to the manufacturer’s instructions with the following modification: 5 μL of CD45 MicroBeads per 10⁷ total cells were added instead of 20 μL. LD columns were used for depletion and the CD45-negative fraction was collected in MACS buffer. Stromal cells from adult thymus were enriched using fluorescence-activated cell sorting (FACS). Blocking was done with human Fc receptor binding inhibitor monoclonal antibody (eBioscience) followed by staining for 20 min using human-specific antibodies against EPCAM (Clone 9C4, BioLegend) and CD45 (Clone HI30, Biolegend). After staining, cells were washed and resuspended in FACS buffer containing DAPI. Cells were sorted on BD FACS Aria II. Pre-gating was first done for live cells based the DAPI stain. The percentages of EPCAM⁺, EPCAM⁻CD45⁻, and CD45⁺ cells in each sample was determined by flow cytometric analysis or by calculating the ratio of cells in epithelial clusters, immune cluster, or other clusters over the total number of cells in the single-cell RNA-seq dataset.