In this study, samples from adult human duodenum and ileum were collected and split for primary tissue single-cell RNA-seq and organoid culture. Human donor tissue was non-inflammed, with terminal ileum samples collected via pinch biopsy during the course of routine colonoscopy, and duodenal samples taken from excess tissue collected during bulk surgical resection. Organoids were cultured and passaged every 6-8 days in Matrigel domes with established media conditions meant to recapitulate the broad diversity of in vivo epithelial cell types (Fujii, M., et al., Cell Stem Cell. 2018). Organoid culture media contained recombinant Noggin, Rspondin-3, FGF2, IGF1, afamin-Wnt3A, in addition to Gastrin and TGF-b inhibitor A83-01 with and without recombinant EGF (E/NR3+F2I1Gi+Af-W3+A83).
Tissue and organoid samples grown in culture over varying periods were dissociated to single cells, and single cell RNA-seq performed via Seq-Well S^3. Analysis was performed following doublet removal with DoubletFinder and data integration (via Seurat v3) across donors and tissues for primary and organoid samples separately. Integrated tissue and organoid datasets were clustered and manually annotated based on marker gene expression. Cells co-expressing ACE2 and TMPRSS2 were identified principally within enterocyte clusters of both tissues and organoids.
Samples from this study were collected in collaboration with Vanessa Mitsialis, MD and Scott Snapper, MD PhD, Boston Children's Hospital.
Note that this is a pre-publication dataset, so in this study we include expression of ACE2 and TMPRSS2 across all cells and expression of all genes in the ACE2+TMPRSS2+ enterocyte subsets.
Note that this dataset was generated for a study unrelated to SARS-CoV-2 and no cells in this study were exposed to this virus.