single - cell suspensions were filtered and forced using a 70 µm nylon cell strainer ( falcon ), followed by centrifugation at 550 g and 4 °c for 5 min and one wash with pbs containing 1 % bovine serum albumin ( bsa; sigma – aldrich ). the single - cell suspensions used for 10x genomics scrna - seq analysis were cleared of red blood cells using a red blood cell lysis buffer ( ebioscience ) followed by live cell counting and loading of 10, 000 cells per lane. we used 10x genomics chromium single cell 3 ′ reagent kits v2 and v3 according to the manufacturers ’ instructions. raw sequencing data were processed using the cell ranger 3. 1. 0 - based hlca pipeline, with the reference genome grch38 and annotation using ensembl98. ambient rna correction was performed with fastcar, using an empty library cutoff of 100 umi and a maximum allowed contamination chance of 0. 05, ignoring the mitochondrial rna. data were merged and processed using seurat, filtering to libraries with > 500 umis and > 200 genes and to the libraries containing the lowest 95 % of mitochondrial rna per sample and < 25 % mitochondrial rna, normalized using sctransform while regressing out variation correlating with the percentage of mitochondrial rna per cell. in general, 15 principal components were used for the clustering, at a resolution of 0. 5 to facilitate manual annotation of the dataset. clusters in the final object that were driven by single donors were removed. raw counts and cell annotations were used as input for the hlca. nasal epithelial samples were collected from healthy volunteers who provided informed consent at northwestern medicine in chicago. the protocol was approved by the northwestern university irb ( stu00214826 ). healthy volunteers were recruited to match a cohort of patients with cystic fibrosis for the ongoing study at northwestern university ( with m. j. as the principal investigator ). in both studies, a. v. m. did not influence participant recruitment and did not introduce biases in sample selection. metadata of the donors ’ sex were based on self - report. briefly, donors were seated and asked to extend their neck. a nasal curette ( rhino - pro; vwr ) was inserted into either nare and gently slid in the direction of posterior to anterior ~ 1 cm along the lateral inferior turbinate. five curettes were obtained per participant. the curette tip was then cut and placed in 2 ml hypothermosol and stored at 4 c until processing. a single - cell suspension was generated using the cold - active dispase protocol reported by deprez et al. and zaragosi and barbry with slight modification. specifically, ethylenediaminetetraacetic acid ( edta ) was omitted and cells were dispersed by pipetting 20 times every 5 min using a 1 ml tip instead of tritr ation using a 21 / 23 g needle. the final concentration of protease from bacillus licheniformis was 10 mg ml. the total digestion time was 30 min. following the wash in 4 ml 0. 5 % bsa in pbs and centrifugation at 400 g for 10 min, cells were resuspended in 0. 5 % bsa in pbs and counted using a nexcelom k2 cellometer with acridine orange / propidium io dide reagent. this protocol typically yields ~ 300 – 500, 000 cells with a viability of > 95 %. the resulting single - cell suspension was then used to generate single - cell libraries following the protocol for 5 ′ v1 ( cg000086 rev m; 10x genomics ) or v2 chemistry ( cg000331 rev a; 10x genomics ). excess cells from two of the samples were pooled together to generate one additional single - cell library. after a quality check, the libraries were pooled and sequenced on a novaseq 6000 instrument. raw sequencing data were processed using the cell ranger 3. 1. 0 pipeline, with the reference genome grch38 and annotation using ensembl98. to assign sample information to cells in the single - cell library prepared from two samples, we ran souporcell version 2. 0 for that library and two libraries that were prepared from these samples separately. we used common genetic variants prepared by the souporcell authors to separate cells into two groups by genotype for each library, and pearson correlation between the identified genotypes across libraries to establish correspondence between genotype and sample. cell annotations were assigned to cell clusters based on expert interpretation of marker genes for each cluster. cell clusters were derived with the seurat version 3. 2 workflow in which samples were normalized with sctransform, 3, 000 hvgs were selected and integrated and clusters were derived from 30 principal components using the louvain algorithm with default parameters. clusters with a low number of umis and high expression of ribosomal or mitochondrial genes were excluded as low quality. raw counts and the thus obtained cell annotations were used as input for the hlca. human lung tissue wabus available for research purposes following ethical approval from hannover medical school ( nr. all patients in this study provided written informed consent for sample collection and data analyses. at hannover medical school, patients with lung cancer were recruited in the course of their operation ( that is, surgical tumor resection was performed according to the ethical vote of the german centre for lung research, ethical vote 7414 and data safety guidelines ). there was no bias in patient recruitment since the samples were collected as fresh native tissue following surgical tumor resection and according to the availability of surplus adjacent nonmalignant lung tissue, which was resected in parallel to the tumor tissue. metadata of the donors ’ sex were based on self - report or reported by medical professionals during consenting. fresh adjacent normal tumor - free lung tissues from patients with non - small cell lung cancer tumors were obtained by the lung research group ( d. jonigk, pathology, hannover medical school ) and processed for single - cell isolation immediately. lung tissue was chopped with a scalpel and scissors and digested using bd tumor dissociation reagent ( bd biosciences ) for 30 min in a 37 °c water bath. the digestion was stopped with 1 % fcs and 2 mm edta in pbs without ca / mg and cells were filtered over a 70 µm cell strainer ( bd falcon ). erythrocytes were removed using a human macsxpress erythrocyte depletion kit ( miltenyi biotec ) and cells were filtered using a 40 µm cell strainer ( bd falcon ). the viability of the cells was assessed microscopically and by flow cytometry using a live / dead fixable yellow dead cell stain kit ( invitrogen ) and was ~ 84 %. the single - cell suspension was processed for scrna - seq and library preparation with the seq - well protocol. library pools with fewer than 100 cells were discarded and merged into one object. the seurat v3. 2 pipeline was used to further analyze the data. genes in fewer than five cells in the dataset, as well as the mitochondrial genes mt - rnr1 and mt - rnr2, were removed. cells with fewer than 200 genes were discarded, whereas cells with < 5 % mitochondrial genes or < 30 % intronic reads were kept for further analysis. the data were log normalized and 2, 000 variable genes were calculated for each sample for integration with seurat ’ s canonical correlation analysis algorithm. the data were scaled, 50 principle components were selected and the data were clustered with 0. 6 resolution. cluster annotation revealed a low - quality cluster that was subsequently removed and the process ( the calculation of variable genes, calculation of 30 principal components, clustering with 0. 4 resolution ) was repeated. raw counts of the cells that passed all filtering were provided as input for the hlca. to accommodate data protection legislation, scrna - seq datasets of healthy lung tissue were shared by dataset generators as raw count matrices, thereby obviating the need to share genetic information. count matrices were generated using varying software ( supplementary table 1 ).