Extended Data Fig. 9: Transition of iRAS cells to iAT2 cells is similar to the primary RAS cell-AT2 transition and is partially regulated by Notch and Wnt.

A) Schematic of time-course of scRNA-seq experiment. B) Integration of entire time course showing cell origin, cell-cycle phase, and gene expression of SCGB3A2 and SFTPC. C) Clustering of complete time course from iRAS  to iAT2 cells development shows multiple clusters within the various time points. D) Trajectory analysis showed multiple putative pseudotemporal orderings (top), and selected curve for further analysis based on termination in day 14 non-mitotic iAT2 cells (bottom). E) Heatmap of iRAS cell to iAT2 cell trajectory displaying genes defining the primary RAS to AT2 cell transition from Extended Data Fig. 5e. F) Expression of a subset of genes identified in primary RAS to AT2 cell transition shown over pseudotime in iRAS  to iAT2 cell transition. G) Canonical airway and alveolar epithelial marker genes expression within the resultant UMAP. H) Flow cytometry of mCherry and eGFP expression in iRAS cells grown in Airway media in the presence or absence of DAPT, and corresponding percent fold change in mCherry expression and MFI. N = 4. I) Flow cytometry of mCherry and eGFP expression of iRAS cells grown in Alveolar media in the presence or absence of DAPT showing percent change in eGFP expression and MFI. J) q-RT-PCR of bulk populations from iRAS cells grown in airway media with or without DAPT. N = 3. K) q-RT-PCR from iRAS cells gown in alveolar media with or without DAPT. N = 3. Quantification of flow cytometry analysis of L) mCherry and M) eGFP positive single cells in culture after iRAS cells were grown in either Airway media or Airway media supplemented with CHIR99021. Data are represented as mean +/- SD and unpaired two-tailed t-tests perfomed. *p < 0.05, **p < 0.01. N) Representative flow cytometry plots (representative of n = 4). O) The heat map of each observed trajectory in panel d is presented with the top trajectory defining genes identified, and cell are ordered by pseudotemporal order on x-axis. All N represent biological replicates

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