A pooled shRNA screen to identify regulators of basal cell fate. (A) Workflow of the pooled shRNA screen. Human airway basal cells were transduced with a lentiviral shRNA library 1 h after they were seeded on Transwell filters at day 0. At day 21, cells were fixed, stained, and sorted into three different groups based on expression of the ciliated cell marker FOXJ1 and the basal cell marker ITGA6. The extracted gDNA from the sorted samples was analyzed for shRNA hairpin counts by NGS. (B) Gene-centric visualization of the log2 fold change and RSA score in the ciliated (ITGA6⁻ FOXJ1⁺) versus secretory (ITGA6⁻ FOXJ1⁻) cells. (C) Ratio of individual shRNA hairpin counts in ciliated/secretory cells from the pooled shRNA screen. KDM8 is shown as an example of a gene whose shRNA barcode counts do not differ between ciliated and secretory cells. (D) In a primary validation, the cells from two independent donors were transduced with four shRNAs for each gene candidate and sorted with FOXJ1 and ITGA6 signals. The ratios of ciliated/secretory cells from all shRNA-treated samples were normalized to those of control cells transduced with shNT. Data were acquired from cells of two donors; mean ± SEM; ***, P < 0.001; Student’s two-tailed t test.