Gene Expression
Nebulas
Gene Expression NebulasSummary: While SARS-CoV-2 infection has pleiotropic and systemic effects in some patients, many others experience milder symptoms. We sought a holistic understanding of the severe/mild distinction in COVID-19 pathology, and its origins. We performed a whole-blood preserving single-cell analysis protocol to integrate contributions from all major cell types including neutrophils, monocytes, platelets, lymphocytes and the contents of serum. Patients with mild COVID-19 disease display a coordinated pattern of interferon-stimulated gene (ISG) expression across every cell population and these cells are systemically absent in patients with severe disease. Severe COVID-19 patients also paradoxically produce very high anti-SARS-CoV-2 antibody titers and have lower viral load as compared to mild disease. Examination of the serum from severe patients demonstrates that they uniquely produce antibodies that functionally block the production of the mild disease-associated ISG-expressing cells, by engaging conserved signaling circuits that dampen cellular responses to interferons. Overzealous and auto-directed antibody responses pit the immune system against itself in many COVID-19 patients and this defines targets for immunotherapies to allow immune systems to provide viral defense
Overall Design: Examination of immune states in patients with and without SARS-Cov2, having mild and severe phenotypes versus healthy controls. (COVID- mild: 6 ; COVID- severe: 5 ; COVID+ mild: 11 ; COVID+ severe: 10 ; healthy controls: 14). M/M = Mild/Moderate
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| Extract Protocol: | Briefly, peripheral blood was collected into EDTA tubes (BD, catalog no. 366643). Whole blood was prepared by treatment of 500µL of peripheral blood with RBC lysis buffer (Roche, 11-814-389-001) according to manufacturer’s procedures. Cells were then counted and 15.000 cells per individual were directly loaded in the ChromiumTM Controller for partitioning single cells into nanoliter-scale Gel Bead-In-Emulsions (GEMs) following manufacturer’s procedures (10x genomics). Some samples were pooled together (at 15,000 cells/ sample) prior to GEM partitioning. |
| Library Construction Protocol: | Single Cell 5’ reagent kit v5.1 was used for reverse transcription, cDNA amplification and library construction of the gene expression libraries (10x Genomics) following the detailed protocol provided by 10x Genomics. |
| 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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