Extended Data Fig. 1: Detailed procedure for the Tn-seq during mucosal colonization.

A. Representative confocal image of (z-slice) showing intact epithe lium after 11 h of infection with the Tn-library. Experiment repeated twice with similar results. B. Exhaustive illustration of experiments and control conditions for the Tn-seq during mucosal colonization. Briefly, an aliquot of Tn-library was grown overnight in LB (starting OD = 0.25) and used as inoculum for infection or a new LB control culture. Infections or growth in the LB control proceeded for ~11 h, followed by sample collection and sequencing. For all details, including ODs used, inoculum sizes, and number of generations measured, see the Methods section. All five samples were sequenced (Tn-library, population used for inoculum, healthy HBE, CF HBE, and LB control). Blue arrows represent the comparisons (#1-3) made using the TRANSIT software to assess the conditional essentiality of genes. In comparison #1, the inoculum was used as the control; in comparison #2 and #3, the “LB control” condition was used as the control. Red arrows represent the two “quality control – QC” comparisons made using the TRANSIT software to check for bias in the inoculum used for infection. Abbreviations: CF, cystic fibrosis; HBE cells, human bronchial epithelial cells; LB, Luria-Bertani broth. The data underlying this figure can be found in the source data available with this manuscript (see Data Availability session).