Gene Expression
Nebulas
Gene Expression NebulasSummary: The SARS-CoV-2 novel coronavirus global pandemic (COVID-19) has led to millions of cases and hundreds of thousands of deaths globally. While older adults appear at high risk for severe disease, hospitalizations and deaths due to SARS-CoV-2 among children have been relatively rare. Integrating single-cell RNA sequencing (scRNA-seq) of developing mouse lung with temporally-resolved immunofluorescence in mouse and human lung tissue, we found expression of SARS-CoV-2 Spike protein primer TMPRSS2 was highest in ciliated cells and type I alveolar epithelial cells (AT1), and TMPRSS2 expression increased with aging in mice and humans. Analysis of autopsy tissue from fatal COVID-19 cases detected SARS-CoV-2 RNA most frequently in ciliated and secretory cells in airway epithelium and AT1 cells in peripheral lung. SARS-CoV-2 RNA was highly colocalized in cells expressing TMPRSS2. Together, these data demonstrate the cellular spectrum infected by SARS-CoV-2 in lung epithelium and suggest that developmental regulation of TMPRSS2 may underlie the relative protection of infants and children from severe respiratory illness
Overall Design: Single Cell RNAseq of dissociated mouse lungs at ages: E18, P0, P7, P14, and P64. Suspensions were enriched for cells that were CD45 negative, Ter119 negative, and viable
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| Extract Protocol: | At the indicated timepoints, lung lobes were harvested, minced, and incubated for 30 minutes at 37°C in dissociation media (RPMI-1640 with 0.7 mg/ml collagenase XI and 30 mg/ml type IV bovine pancreatic DNase). After incubation, lobes were passed through a wide bore pipet tip and filtered through a 40 μm filter. Single cell lung suspension was then counted, aliquoted, and blocked with CD-32 Fc block (BD cat #553142) for 20 minutes on ice. After 2% FBS staining buffer wash, cells were incubated with the conjugated primary antibodies anti-CD45 (BD cat # 559864) and anti-Ter119 (BD cat# 116211). In the same manner, fluorescence minus one controls were blocked and stained with the appropriate antibody controls. Cells from individual mice were then incubated with identifiable hashtags, resuspended in staining buffer, and treated with PI viability dye. CD45 negative, Ter119 negative, viable cells were collected by fluorescence associated cell sorting using a 70 μm nozzle on a 4-laser FACSAria III Cell Sorter. Both single and fluorescence-minus-one controls were used for compensation. |
| Library Construction Protocol: | ScRNA-seq libraries were generated using the 10X Chromium platform 5’ library preparation kits (10X Genomics) following the manufacturer's recommendations and targeting 10,000 - 20,000 cells per sample. |
| Molecule Type: | Poly(A)+ RNA |
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| Library Layout: | PAIRED |
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| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| Strand-Specific: | - |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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