The single-cell suspension was applied to the 10× Genomics Chromium platform (San Francisco, CA) to capture and barcode cells, as described in the manufacturer’s protocol. Libraries were constructed using the Single Cell 3′ Reagent Kit (v2 Chemistry). The completed libraries were then sequenced using HiSeq 2500 (Illumina, San Diego, CA) running in Rapid Mode. Each sample was loaded onto two lanes of a Rapid v2 flow cell.

Raw data from 10× Genomics were demultiplexed and converted to a fastq file using cellRanger (RRID:SCR_017344; v2.1.1) mkfastq. Reads from the same library sequenced in different flow cells (technical replicates) were combined and aligned to the mm10 genome reference using cellRanger count. Summary data for statistical mapping profiles are presented in Supplementary file 2a. The gene expression profiles for cells from the three biological replicates of the IH group were combined with cellRanger aggr and were run an unsupervised analysis using the software Iterative Clustering and Guide-gene Selection (ICGS) versions 2 (AltAnalyze version 2.1.2) to generate reference clusters using the program defaults with Euclidean clustering (DePasquale et al., 2019). ICGS2 grouped 12,324 cells into 25 reference clusters based on the expression profiles of 1480 selected marker genes (Supplementary file 2b). All cells from control and IH groups were then aligned to these 25 reference clusters using cellHarmony (DePasquale et al., 2019). Uniform Manifold Approximation and Projection (UMAP) calculation was run using integrated function in AltAnalyze (RRID:SCR_002951) -v2.1.2 with default parameters. For annotating the 25 reference clusters into known lung cell types, we prepared a comprehensive marker gene list for known lung cell types. The sources of this marker gene list included information from the Mouse Cell Atlas, ToppGene, and Lung Gene Expression Analysis (LGEA) (Tabula Muris Consortium et al., 2018; Chen et al., 2009; Du et al., 2017). Additionally, we manually collected cell marker genes from published scRNA-seq studies performed in mouse or human lung (Zilionis et al., 2019; Guo et al., 2019). One-tailed Fisher’s exact test was used to perform enrichment analysis between marker genes for each cluster and the curated reference markers of known lung cell types. Each cluster was manually assigned to a specific cell type based on the known cell type with the lowest BH (Benjamini and Hochberg, 1995) adjusted p-value (GO-Elite software) (Zambon et al., 2012). Those clusters corresponding to the same annotated cell type were manually joined as one cell type for downstream analyses (e.g. endothelial corresponding to four clusters). This process reduced the 25 reference clusters into 19 cell types (Supplementary file 2c). For testing the cell-type composition difference of mouse lung between experimental and control groups, a centered log ratio transformation was performed on the percentage of each cell type before applying the t-test (two tailed). The statistical p-value from the t-test was adjusted with the BH method.

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