Extended Data Fig. 6: Lineage tracing using pulse-seq.

a, Schematic of the pulse-seq experimental design. b, Post hoc cluster annotation by known cell type markers. t-SNE of 66,265 scRNA-seq profiles (points) from pulse-seq, coloured by the expression (log₂(TPM+1)) of single marker genes for a particular cell type or cell-cycle score (bottom right) c, Pulse-seq lineage-labelled fraction of various cell populations over time. Linear quantile regression fits (trendline) to the fraction of lineage-labelled cells of each type (n = 3 mice per time point, dots) as a function of the number of days after tamoxifen-induced labelling. β, estimated regression coefficient, interpreted as daily rate of new lineage-labelled cells; p, P value for the significance of the relationship, Wald test. As expected, goblet and ciliated cells are labelled more slowly than club cells (Fig. 3d). d, Labelled fraction of basal cells is unchanged during pulse-seq time course, as expected. Estimated fraction (%) of cells of each type that are positive for the fluorescent lineage label (by FACS) in each of n = 3 mice (points) per time point. P values, LRT; error bars, 95% CI. e, Proportion of basal cell lineage-labelled tuft cells at day 0 (0%; n = 2 mice, dots) and day 30 (22.9%, 95% CI [0.17, 0.30]; bars, estimated proportions; n = 3 mice). Error bars, 95% CI; P values, LRT. f–h, Conventional Scgb1a1 (CC10) lineage trace of rare epithelial types shows minimal contribution to rare cell lineages. Fraction of Scb1a1 labelled (club cell trace) cells (%) of Gnat3⁺ tuft cells (f) at day 0 (n = 3 mice; 0.6%, 95% CI [0.00, 0.04]) and day 30 (n = 2 mice; 6.3%, 95% CI [0.04, 0.11]), EGFP(Foxi1)⁺ ionocytes at day 30 (n = 2 mice; 2.9%, 95% CI [0.01, 0.11]) (g), and Chga⁺ neuroendocrine cells at day 0 (n = 2 mice; 2.5%, 95% CI [0.01, 0.08]) and day 30 (n = 2 mice; 2.6%, 95% CI [0.01, 0.08]) (h) after club cell lineage labelling. P values, LRT; error bars, 95% CI.