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| Status |
Public on Jun 27, 2021 |
| Title |
Virus+RDV-24hpi-Rep1-VeroE6-PolyA |
| Sample type |
SRA |
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| Source name |
Vero E6 cells
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| Organisms |
Chlorocebus sabaeus; Severe acute respiratory syndrome coronavirus 2 |
| Characteristics |
cell line: Vero E6
treatment: SARS-CoV-2 + Remdesivir
time: 24hpi
rna population: PolyA RNA
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| 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.
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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.
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| Extracted molecule |
polyA RNA |
| Extraction 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)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The raw data was quality controled with fastp, with parameters "-n 10 -q 35 -w 8 --detect_adapter_for_pe".
The sequenced reads of Vero-E6 cell lines were aligned to the ChlSab1.1 reference genomes respectively by using STAR 2.7.1a [35]. Reference genome was downloaded from htps://www.ensembl.org/info/data/ftp/index.html.
Unmapped reads were output as fastq files and then aligned to SARS-COV-2 genome, which was downloaded from https://www.ncbi.nlm.nih.gov/nuccore/MT123290.1?report=fasta, by STAR
Picard toolkit v2.22.6 (“Picard Toolkit.” 2019) with option “MarkDuplicates” to mark the duplicated reads in sorted bam file that mapped to virus genome, then we used samtools v1.9 with option “-F 1024” to remove the duplicated reads. Cigars are extracted from bam files and results are listed in the Processed files ended with .cigar_jumps_no_dup.txt.
Bedtools v2.28.0 [37] with option “genomecov -strand +/-” was used to calculate coverage of reads mapped to both sense and anti-sense strand genome for Ribozero sequencing. (Processed files ended with jumps.txt )
Genome_build: Chlorocebus sabaeus: htps://www.ensembl.org/info/data/ftp/index.html. SARS-CoV-2: https://www.ncbi.nlm.nih.gov/nuccore/MT123290.1?report=fasta
Supplementary_files_format_and_content: txt format. The processed files contain position of junciton sites on both strands and cigar of reads mapped to SARS-CoV-2 genome.
Supplementary_files_format_and_content: *unmapped.cigar_jumps_no_dup.txt: each line starts with a number recording the #[StartMappingPostion], followed by a capital 't' to separate it with the [cigars extracted from bam files, eg. 50M3000N100M].
Supplementary_files_format_and_content: *unmapped.jumps_no_dup.txt: the j5 junction site, j3 junction site and read counts seperated by a capital 't'.
Supplementary_files_format_and_content: *FKDL*.cigar_jumps.txt: the strand flag, mapping start position of paired reads and mapping results (cigar from the bam files) are seperated by a space. [header: strand, StartPos, cigar]
Supplementary_files_format_and_content: *FKDL*.jumps.txt: header: [j5-junction-site j3-junction-site Strand ReadsCount]
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| Submission date |
Nov 02, 2020 |
| Last update date |
Jun 27, 2021 |
| Contact name |
Yan Zhao |
| E-mail(s) |
11962001@mail.sustech.edu.cn
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| Organization name |
Southern University of Science and Technology
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| Department |
BIology
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| Street address |
No. 1088,Xueyuandadao
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| City |
Shenzhen |
| ZIP/Postal code |
518055 |
| Country |
China |
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| Platform ID |
GPL29334 |
| Series (1) |
| GSE160668 |
Analysis on Replication and Transcription of SARS-CoV-2 and inhition effect of Remdesivir |
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| Relations |
| BioSample |
SAMN16628285 |
| SRA |
SRX9419889 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4876680_VA12_unmapped.cigar_jumps_no_dup.txt.gz |
144.8 Kb |
(ftp)(http) |
TXT |
| GSM4876680_VA12_unmapped.jumps_no_dup.txt.gz |
24.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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