Gene Expression
Nebulas
Gene Expression NebulasSummary: The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNAseq to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of cell types are susceptible to infection, ciliated cells are a predominant cell target for SARS-CoV-2. Remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. We also found that heavily infected epithelial cells demonstrate impaired IFN signaling and express abundant IL-6, a potential mediator of COVID-19 pathogenesis.
Overall Design: Basal layer of medium was replaced with infection media (DMEM with 2% FBS, 100 U/mL penicillin and streptomycin, 10 mM HEPES, 0.1 mM nonessential amino acids, 4 mM L-glutamate, and 1mM sodium pyruvate). Wells were left untreated, or treated with 100 ng of IFN-b or 0.1 mM remdesivir in the basal compartment for 1 hr prior to infection. For infection, SARS-CoV-2 strain 2019-nCoV/USA_WA1/2020 (provided by World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch) and icSARS-CoV-2-mNG, were added directly to the apical layer of cells for 1 hr at 37掳C with rocking every 10 min. The virus was removed from the apical compartment and cells were incubated at 37掳C until harvest.
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| Growth Protocol: | nHTBE cells (Lonza Bioscience, CC-2540S) harvested from healthy individuals were expanded using PneumacultEx Plus (Stem Cell Technologies) and seeded on 24 well hanging inserts, 0.4 pore (Corning, USA) and were maintained at ALI for 21+ days in a PneumacultALI growth medium (Stem Cell Technologies) at 37掳C/5% CO2 in a humidified incubator. Cells were used 21+ days post ALI for experiments. |
| Treatment Protocol: | Basal layer of medium was replaced with infection media (DMEM with 2% FBS, 100 U/mL penicillin and streptomycin, 10 mM HEPES, 0.1 mM nonessential amino acids, 4 mM L-glutamate, and 1mM sodium pyruvate). Wells were left untreated, or treated with 100 ng of IFNor 0.1 remdesivir in the basal compartment for 1 hr prior to infection. For infection, SARS-CoV-2 strain 2019-nCoV/USA_WA1/2020 (provided by World Reference Center for Emerging Viruses and Arboviruses at the University of Texas Medical Branch) and icSARS-CoV-2-mNG, were added directly to the apical layer of cells for 1 hr at 37 with rocking every 10 min. The virus was removed from the apical compartment and cells were incubated at 37 until harvest. |
| Extract Protocol: | Cells were washed and trypsinzed using ReagentPackSubculture Reagents (Lonza Bioscience). Single cell suspensions were washed with PBS + 5% FBS, resuspended in 200 L PBS + 5% FBS, passed through a 70 filter, and placed on ice. Cells were counted using trypan blue exclusion on a Luna II (Logos Biosystems). The appropriate numbers of cells to achieve a targeted cell input of 5,000 cells per condition were used to generate the GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single Cell 3Gel beads v3.1 kit (10X Genomics, Pleasanton, CA, USA) was used to create GEMs following manufacturer instruction. All samples and reagents were prepared and loaded into the chip and ran in the Chromium Controller for GEM generation and barcoding. |
| Library Construction Protocol: | GEMs generated were used for cDNA synthesis and library preparation using the Chromium Single Cell 3 Library Kit v3.1 (10X Genomics) following the manufacturer instruction.; - |
| Molecule Type: | Poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| 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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