Gene Expression
Nebulas
Gene Expression NebulasSummary: Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection
Overall Design: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control SARS-CoV-2 infection remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively which cellular and viral RBPs are involved in SARS-CoV-2 infection. We reveal that the cellular RNA-bound proteome is remodelled upon SARS-CoV-2 infection, having widespread effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.
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| Growth Protocol: | Cells were maintained in DMEM supplemented with 10%FBS |
| Treatment Protocol: | Knockdown was induced by treatment with 1ug/ml Doxycyclin; Cells were seeded into 2.5x10^5/well into 24well plates and infected at MOI=0.1 for 24hpi |
| Extract Protocol: | 24hpi cells were detached and lysed in Trizol LS, total RNA extraction was performed following manufacturers recommendation RNA sequencing libraries were prepared using the Illumina Total RNA Prep with Ribo-Zero Plus library kit (Cat# 20040525) according to manufacturers guidelines. Briefly, 100ng of total RNA was first depleted of the abundant ribosomal RNA present in the samples by rRNA targeted DNA probe capture followed by enzymatic digestion. Samples were then purified by Beckman Coulter RNAClean XP beads (Cat #A63987). Obtained rRNA-depleted RNA was fragmented, reverse transcribed, converted to dsDNA, end repaired and A-tailed. The A-tailed DNA fragments were ligated to anchors allowing for PCR amplification with Illumina dual indexing primers (Cat#20040553) |
| Library Construction Protocol: | Libraries were pooled in equimolar concentrations and sequenced on an Illumina NextSeq 500 sequencer using a high-output cartridge (Cat# 20024907), generating single 150bp long reads. |
| Molecule Type: | rRNA- RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | 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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