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Fig. 1 | BMC Biology

Fig. 1

From: Harnessing single-cell genomics to improve the physiological fidelity of organoid-derived cell types

Fig. 1

Transcriptional benchmarking of in vitro Paneth cells (PCs) to in vivo. a Schematic of intestinal epithelial cell isolation from terminal ileum for unbiased identification of in vivo PC signature genes, and system for intestinal stem cell (ISC) enrichment to characterize in vitro PCs, via high-throughput scRNA-seq. b Marker gene overlay for binned count-based expression level (log(scaled UMI + 1)) of Lyz1, a canonical PC marker gene, on a tSNE (t-stochastic neighbor embedding) plot of 7667 small intestinal epithelial cells isolated from the terminal ileum; receiver operating characteristic (ROC)-test area under the curve (AUC) = 0.995, n = 2 mice, independent experiments (Additional file 1: Table S1). c Violin plot for the count-based expression level (log(scaled UMI + 1)) of Lyz1 across clusters identified through shared nearest neighbor (SNN) analysis (see Methods) over small intestinal epithelial cells; n = 196 cells in cluster 11, 7667 cells in total. d A tSNE plot of 2513 cells, with clusters identified through SNN (Additional file 1: Table S1 for full gene lists with ROC-test AUC > 0.60) from conventional ENR organoids; n = 6 wells of ENR organoids. e Marker gene overlay for binned count-based expression level (log(scaled UMI + 1)) of Lyz1 on a tSNE plot from; ROC-test AUC = 0.856. f Violin plot of expression contribution to a cell’s transcriptome of PC genes across ENR organoid clusters from (d) (In vivo PC gene list AUC > 0.65, Additional file 1: Table S1); effect size 0.721, ENR-4 vs. all ENR, *t test p < 2.2 × 10−16. g Row-normalized heatmap of top differentially expressed genes using bimodal test over single-cells from the top 200 PC-like cells from ENR-4 and the 196 in vivo PCs (cluster 11, from (c)); *bimodal test, all displayed genes p < 1.89 × 10−16 or less with Bonferroni correction. h Violin plots for the count-based expression level (log(scaled UMI + 1)) of Lyz1, Ang4, and Defa3 in ENR and in vivo PCs; *bimodal test, all p < 2.92 × 10−37 or less with Bonferroni correction. i Violin plot of expression contribution to a cell’s transcriptome of PC genes (effect size 1.25, InVivo vs. ENR, *t test p < 2.2 × 10−16), Wnt pathway (effect size 0.559, InVivo vs. ENR, *t test p < 2.035 × 10−8) and Notch pathway (effect size −0.500, InVivo vs. ENR, *t test p < 5.25 × 10−7) genes (see Additional file 2: Table S2 for gene lists)

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