Gene Expression
Nebulas
Gene Expression NebulasSummary: SARS-CoV-2, a betacoronavirus with a positive-sense, single-stranded RNA genome, caused the COVID-19 pandemic with unprecedent health and socio-economic crisis. Although its general sense mRNA architecture was reported, that of its negative strand was unexplored. We combined poly(A)-mRNA and ribosomal RNA-depleted sequencing to delineate several fine features of both RNA strands. Together with the transcriptome data under the perturbation of anti-viral drug Remdesivir, this project opens new sights into SARS-CoV-2 replication mechanism and may facilitate the anti-viral drug design.
Overall Design: Overall 10 samples are analyzed, six of which are Poly(A) RNA-seq and four are Ribozero RNA-seq samples. Poly(A) RNA-seq samples contain two treatment conditions with triplicates. Ribozero RNA-seq samples contain two treatment conditions with duplicates.
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| Growth Protocol: | Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) at 37 °C in a humidified atmosphere of 5% CO2. |
| Treatment Protocol: | For virus infection, Vero E6 cells, with 90% confluency in 12-well plates, were inoculated with the SARS-CoV-2 virus at a MOI of 0.05. One hour after incubation at 37 °C, cells were washed three times with phosphate buffered saline (PBS) followed by 24-hours.Remdesivir (Cat. No. HY-104077) was purchased from MedChemExpress (Monmouth Junction, NJ). The SARS-CoV-2 strains used in this research were isolated from COVID-19 patients in Guangzhou (Accession numbers: MT123290 ), and passaged on Vero E6. incubation in the fresh normal culture medium with or without Remdesivir at 10 uM of final concentration. |
| Extract Protocol: | Cultured cells were washed once with PBS before adding TRIzol (Vazyme, Cat no. R401-01). Total RNA extracted according to the manufacturer’s instructions. RNA was eluted in 20 μl RNase-free water. Purified total RNAs from non-infected and SARS-CoV-2-infected Vero cells were reverse transcribed using the RT SuperMix Reagent Kit with gDNA Eraser (Vazyme, Cat no. R223-01). Briefly, 1 μg total RNA was firstly digested with gDNA eraser to remove contaminated DNA and then the first-strand cDNA was synthesized in 20 μl reaction with Oligo (dT) or forward and reverse PCR primer for negative-strand and positive-strand specific reverse transcription, respectively. Finally, 2 μl ten times diluted cDNA was used as template for quantitative PCR. RT-qPCR was performed on CFX96 Real-time PCR system (Bio-Rad) with the SYBR Green Master Mix (Yeasen, Cat no. 11201ES03) |
| Library Construction Protocol: | - |
| Molecule Type: | polyA(+) RNA; rRNA- RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward; Reverse |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NovaSeq 6000 |
| 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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