mRNA-seq of large airway basal cell, small airway basal cell and alveolar type2 cells from normal patients

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 cellular entry, and their expression may shed light on viral tropism and impact across the body. We assess the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial–macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of coronavirus disease 2019, and our work highlights putative molecular pathways for therapeutic intervention. Single-cell meta-analysis not included in this submission. Overall design: Human lung tissue was received from New England Donor Services under the Massachusetts General Hospital approved institutional review board protocol. Tissue was used from three individual patients with no history of lung disease or smoking. Primary bronchus and a piece of right lung lobe were manually dissected out. Single cells were dissociated in HBSS media (sigma, 55021C) containing collagenase (225 units/ml), dispase (2.5 units/ml), elastase (2 units/ml), pronase (1mg/ml), DNAse (1 unit/ml) and Y-27632 (5uM) inhibitor. Cell suspension was treated with ACK lysis buffer (thermofisher scientific A1049201) for 2 minutes on ice to remove red blood cells. Large airway basal cells were isolated from primary bronchus tissue while small airway basal cells and alveolar type 2 (AT2) cells were isolated from lung lobe tissue. Basal cells from both tissue suspensions were isolated using the anti-human CD271 MicroBeads kit (Miltenyl Biotech, 130-099-023) following manufacturers protocol. AT2 cells were isolated with Anti HT2-280 antibody (Terrace Biotech, TB-27AHT2-280) and anti-Mouse IgM MicroBeads kit (Miltenyl Biotech, 130-047-301) following manufacturers protocol. For mRNA-Seq sample preparation, the total RNA was isolated using Trizol reagent (Thermofischer Scientific, 15596026) following the manufacturer's instructions. Quality and quantity of total RNA was assayed using Nanodrop and Agilent Bioanalyzer. Further, mRNA-seq libraries were prepared using the TrueSeq protocol from Illumina. For mRNA-Seq analysis, the raw fastq files were mapped to the human genome using STAR (Version 20201). The output files were processed through Cufflinks (Version 2.2.1.3)) and Cuffdiff (Version 2.2.1.6) to conduct differential gene expression analysis.