Gene Expression
Nebulas
Gene Expression NebulasSummary: SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in Type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factors p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication.
Overall Design: (1) Independent biological triplicates of transformed lung alveolar (A549) cells transduced with a vector expressing human ACE2 infected with SARS-CoV-2 (USA-WA1/2020, MOI: 2) for 2/4/6/9/12/18/24 hours. Samples were examined by mRNA-seq. (2) Independent biological triplicates of transformed lung alveolar (A549) cells transduced with a vector expressing human ACE2 infected with SARS-CoV-2 (USA-WA1/2020, MOI: 0.01) for 24 hours. Samples were examined by scRNA-seq. (3) Independent biological triplicates of transformed lung alveolar (A549) cells transduced with a vector expressing human ACE2 infected with SARS-CoV-2 (USA-WA1/2020, MOI: 0.1) for 24 hours. Samples were examined by ATAC-seq.
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| Growth Protocol: | - |
| Treatment Protocol: | Cells were infected with SARS-CoV-2 (USA-WA1/2020) at the indicated MOI and for the indicated amount of time. Alternatively, mock infected cells were treated with |
| Extract Protocol: | Cells were homogenized in TRIzol reagent and total RNA was extracted using the Direct-zol RNA Miniprep kit according to the manufacturer's instructions; Cells were trypsinized with 0.25% trypsin and collected as a single cells suspension. Cells were then washed twice with ice cold 1x PBS and filtered using a 40 μm Flowmi cell strainer (Bel-Art Scienceware). Cell count and viability were determined using trypan blue stain and a Countess II automatic cell counter (Thermfisher Scientific). Based on this cell count, a target cell input volume of 3,000 cells was loaded into a Chromium Controller using Chromium Next Gem (Gel Bead-In Emulsion) Single Cell 5’ Library & Gel Bead Kit v1.1 (10x Genomics) according to manufacturer’s instructions. |
| Library Construction Protocol: | TruSeq Stranded mRNA Library Prep Kit (Illumina); Chromium Single Cell 5’ Library Kit v1.1 (10x Genomics) |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Forward; - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Specific; - |