Fig. 1

Transcriptomic analysis of organoid cells isolated from TRACER reveals heterogeneity that is not exclusively dependent on layer of origin. (A) Schematic of the experimental workflow. (B) UMAP plot of organoid cells isolated from all layers of the TRACER construct. Seven organoid cell clusters were identified. (C) UMAP plot of organoid cells isolated from all layers of TRACER coloured by layer of origin reveals cluster identity was determined by gene expression patterns rather than exclusively layer of origin. (D) Heatmap of 10 most differentially expressed genes for each of the seven single-cell clusters. Cluster identity and layer of origin are shown in the coloured bars. Heatmap was made using the Scillus package v 0.5.0. (E) Gene set enrichment analysis by cluster using gene sets compiled from multiple databases indicating gene sets that were significantly enriched (adjusted p-value < 0.05) across databases. The maximum normalized enrichment score (NES) for the reoccurring gene sets is represented. Gray boxes indicate no significant enrichment. A summary of the gene sets and their source database is provided in Table S1. (F) Visualization of a representative gene for each cluster-defining gene set supports cluster annotation [DDIT3 – Unfolded Protein Response, Hypoxia (Hypoxia Response); ITGB1 – EMT, Integrin Signalling (EMT); MKI67 – Cell proliferation, E2F targets (Cell Cycle); PDK4 – Oxidative Phosphorylation (Aerobic Processes); SF3A2 – mRNA Processing (RNA Processing); TNFAIP3 – TNFα signalling (Hypoxia & TNF-α Signalling); IRF1 – Interferon signalling (Inflammatory Response)].