Loupe cell browser

Immediately postsorting, CD45⁻ DAPI⁻ EpCAM⁺total TECs were run on the 10× Chromium and then single‐cell RNA‐seq libraries were generated using the Chromium Single Cell 3′ Reagent Kit (10× Genomics) by the core facility at the Embryology Department of Carnegie Institute for Science. Briefly, TEC single‐cell suspension (~1,000 cells per 1 µl PBS) was mixed thoroughly with Single Cell 3′ gel beads and partitioning oil into a Single Cell 3′ Chip (10× genomics) following the recommended protocol for the Chromium Single Cell 3′ Reagent Kit (v2 Chemistry). RNA transcripts of single cells were uniquely barcoded and reverse‐transcribed within the individual droplets. cDNA molecules were then pre‐amplified and pooled together followed by the final library construction. Libraries were sequenced by paired‐end 150‐bp reads on Illumina NextSeq 500. Postprocessing and quality control were performed by the same genomics core facility using the 10× Cell Ranger package (V2.1.1, 10× Genomics) as described by Zheng et al. (2017). Reads were aligned to mm10 reference assembly (v1.2.0, 10× Genomics). After cell demultiplexing and read counting, all three samples were combined using the cellranger aggr pipeline (Zheng et al., 2017). Cell visualization was done using the Loupe Cell Browser (10× Genomics).