the libraries were prepared as described in deprez et al. and yielded an average of 61, 000 ± 11, 000 cells per sample, with a viability above 95 %. the single - cell suspension was used to generate single - cell libraries following the v3. 1 protocol for 3 ′ chemistry from 10x genomics ( cg000204 ). sequencing was performed on a nextseq 500 / 550 sequencer ( illumina ). raw sequencing data were processed using the cell ranger 6. 0. 0 pipeline, with the reference genome grch38 and annotation using ensembl98. for each sample, cells with fewer than 200 transcripts or more than 40, 000 transcripts were filtered out, as well as genes expressed in fewer than three cells. normalization and log transformation were done using the standard scanpy pipeline. principal component analysis ( pca ) was performed on 1, 000 highly variable genes ( hvgs ) to compute 50 principal components, and the louvain algorithm was used for clustering. these clusters were then annotate d by hand for each sample. raw counts and the thus obtained cell annotations were used as input for the hlca. tumor - free, uninvolved lung samples ( peritumor tissues ) were obtained during tumor resections at the lung specialist clinic asklepios fachkliniken munchen - gauting and accessed through the bioarchive of the comprehensive pneumology center in munich. the study was approved by the local ethics committee of the ludwig maximilian university of munich ( ek 333 - 10 and 382 - 10 ), and written informed consent was obtained from all patients. all fresh tissues from patients in a given time frame without any specific selection criteria were included, and only patients with obvious chronic lung disease as comorbidity based on their lung function parameters before tumor resection were excluded. metadata of the donors ’ sex were based on self - report. single - cell suspensions for scrna - seq were generated as previously described. in brief, lung tissue samples were cut into smaller pieces, washed with phosphate - buffered saline ( pbs ) and enzymatically digested using an enzyme mix compose d of dispase, collagenase, elastase and dnase for 45 min at 37 °c while shaking. after inactivating the enzymatic activity with 10 % fetal calf serum ( fcs ) / pbs, dissociated cells were passed through a 70 µm cell strainer, pelleted by centrifugation ( 300 g; 5 min ) and subjected to red blood cell lysis. after stopping the lysis with 10 % fcs / pbs, the cell suspension was passed through a 30 µm strainer and pelleted. cells were resuspended in 10 % fcs / pbs, assessed for viability and counted using a neubauer hematocytometer. the cell concentration was adjusted to 1, 000 cells per µl and ~ 16, 000 cells were loaded on a 10x genomics chip g with chromium single cell 3 ′ v3. 1 gel beads and reagents ( 3 ′ gex v3. 1; 10x genomics ). libraries were prepared according to the manufacturer ’ s protocol ( cg000204 _ revd; 10× genomics ). after a quality check, scrna - seq libraries were pooled and sequenced on a novaseq 6000 instrument. the generation of count matrices was performed using the cell ranger computational pipeline ( v3. 1. 0; star v2. 5. 3a ). the reads were aligned to the grch38 human reference genome ( grch38; ensembl99 ). downstream analysis was performed using the scanpy package ( version 1. 8. 0 ). we assessed the quality of our libraries and excluded barcodes with fewer than 300 genes detected, while retaining those with a number of transcripts between 500 and 30, 000. furthermore, cells with a high proportion ( > 15 % ) of transcript counts derived from mitochondrial - encoded genes were removed. genes were considered if they were expressed in at least five cells. raw counts of cells that passed filtering were used as input for the hlca. all postmortem human donor lung samples were obtained from brindl, supported by the nhlbi lungmap human tissue core housed at the university of rochester. consent, tissue acquisition and storage protocols can be found on the repository ’ s website ( brindl. urmc. rochester. edu / ). data were collected as part of the human biomol ecular atlas program ( hubmap ). metadata of the donor ’ s sex were based on self - report. for isolation of single nuclei, ten cryosections ( 40 µm thickness ) from o. c. t. - embedded tissue blocks stored at −80 °c were shipped on dry ice and processed according to a published protocol. single-nucleus rna - seq was completed using 10x chromium single cell 3 ’ reagent kits v3, according to a published protocol. raw sequencing data were processed using the 10x cell ranger v3 pipeline and the grch38 reference genome. for downstream analysis, mitochondrial transcripts and doublets identified by doubletdetection version 2. 4. 0 were removed. samples were then combined and cell barcodes were filtered based on the genes detected ( > 200 and < 7, 500 ) and the gene unique molecular identifier ( umi ) ratio ( gene. vs. molecule. cell. filter function ) using pagoda2 ( github. com / hms - dbmi / pagoda2 ). also using pagoda2 for clustering, counts were normalized to total counts per nucleus. for batch correction, gene expression was scaled to dataset average expression. after variance normalization, all significantly variant genes ( n = 4, 519 ) were used for pca. clustering was done at different k values ( 50, 100 or 200 ) using the top 50 principal components and the infomap community detection algorithm. then, principal component and cluster annotations were imported into seurat version 4. 0. 0. differentially expressed genes for all clusters were generated for each k resolution using seurat findallmarkers ( only. pos = true, max. cells. per. ident = 1000, logfc. threshold = 0. 25, min. pct = 0. 25 ). clusters were manually annotate d based on distinc t differentially expressed marker genes. raw counts and the thus obtained cell annotations were used as input for the hlca. these data were a combination of published and unpublished data. in both cases, healthy volunteers were recruited for bronchoscopy at the university medical center in groningen after giving informed consent and according to the protocol approved by the irb ( abr number nl69765. 042. 19 ). inclusion criteria and tissue processing were performed as previously described. in short, all donors were 20 – 65 years old and had a history of smoking < 10 pack - years. metadata of the donors ’ sex were based on self - report. to exclude respiratory disease, the following criteria were used: absent history of asthma or copd; no use of asthma - or copd - related medication; a negative provocation test ( concentration of methacholine that provokes a 20 % decrease in the forced expiratory volume in 1 s ( fev 1 ) > 8 mg ml ); no airflow obstruction ( fev 1 / forced vital capacity ≥ 70 % ); and an absence of lung function impairment ( that is, fev 1 ≥ 80 % predicted ). all donors underwent a bronchoscopy under sedation using a standar diz ed protocol. nasal brushes were obtained from the lateral inferior turbinate in a subset of the volunteers immediately before bronchoscopy using a cyto-pak cytosoft nasal brush ( medical packaging corporation ). six macroscopically adequate endobronchial biopsies were collected for this study, located between the third and sixth generation of the right lower and middle lobe. bronchial brushes were obtained from a different airway at similar anatomical locations using a cellebrity bronchial brush ( boston scientific ). extracted biopsies and bronchial and nasal brushes were processed directly, with a maximum of 1 h delay. bronchial biopsies were chopped biopsies using a single - edge razor blade. a single - cell solution was obtained by tissue digestion using 1 mg ml collagenase d and 0. 1 mg ml dnase i ( roche ) in hanks ’ balanced salt solution ( lonza ) at 37 °c for 1 h with gentle agitation for both nasal brushes and bronchial biopsies.