Lung distal epithelium ChiaRed and WT epithelial cells

Using a method that enriches for alveolar epithelial cells, we compared distal epithelial cells from wild-type (WT) mice with AMCase reporter (ChiaRed; CR)-expressing epithelial cells from heterozygous CR mice by scRNAseq to further verify the cellular identity of AMCase-expressing cells. In the steady-state, EpCAM+ cells from WT mice were primarily comprised of mature AT2 (93%) and Tm4sf1-expressing alveolar epithelial progenitor cells (AEP; ~6%); AMCase-expressing CR+ cells matched the transcriptional profile of mature AT2s, as expected based on prior studies. In contrast to AT2s, AEPs were comparatively enriched for transcripts marking transitional alveolar epithelial cell states (Krt8, Krt19, Cldn4, Cdkn1a) that expand in pathological settings such as severe SARS-CoV-2 infection and pulmonary fibrosis. In addition, AEPs and AT2s differentially expressed Epcam, Cdh1, and H2-Ab1, encoding cell surface markers EpCAM, E-cadherin, and MHC-II, respectively, which were used to distinguish these populations by flow cytometry.

Methods
Single-cell RNA sequencing (scRNAseq) was performed with WT (DAPICD45- EpCAM+) or CR+ (DAPICD45- EpCAM+CR+) epithelial cells, sorted into ice-cold 0.5% BSA in PBS and processed through the Chromium Single Cell 3' v2 Library Kit (10X Genomics) per the manufacturer’s protocol. Single-cell libraries from 10,000 cells per sample were sequenced with standard Illumina sequencing primers on an Illumina HiSeq 4000, using paired-end sequencing with single indexing, in which read 1 was 26 cycles and read 2 was 98 cycles. The resulting bcl files were de-multiplexed using bcl2fastq2.1.7v, and the resultant paired-end fastq files were aligned to the mm10 transcriptome (ftp://ftp.ensembl.org/pub/release84/fasta/mus_musculus/dna/Mus_musculus.GRCm38.dna.primary_assembly.fa.gz and ftp://ftp.ensembl.org/pub/release-84/gtf/mus_musculus/Mus_musculus.GRCm38.84.gtf.gz) using STAR aligner in the Cellranger toolkit (10X Genomics).