Bronchial brushings were treated with 0.25% trypsin/EDTA for 20 min and stained for sorting using a BD FACSAria II. Gating based on forward scatter height (FSC-H) versus forward scatter area (FSC-A) was applied to sort only singlet events (fig. S1A). Dead cells (LIVE/DEAD Fixable Aqua Dead Cell Stain, Thermo Fisher; L34957) and red blood cells expressing GYPA/B (fig. S1B) on their surface [allophycocyanin (APC) anti-CD235ab; BioLegend, 306607] were stained and excluded. ALCAM+ epithelial cells [phycoerythrin (PE) anti-CD166; BioLegend, 343903] and CD45+ WBCs (APC-Cy7 anti-CD45; BD, 561863) were stained (fig. S1C) and sorted into 96-well polymerase chain reaction (PCR) plates containing lysis buffer [0.2% Triton X-100, 2.5% RNaseOUT (Thermo Fisher; 10777019)] compatible with downstream RNA library preparation. In each 96-well PCR plate for each subject, we sorted 84 ALCAM+ cells and 11 CD45+ cells and maintained one empty well as a negative control. The plates were frozen on dry ice and stored at −80°C until preparation for sequencing.

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