|
| Status |
Public on Sep 25, 2020 |
| Title |
HK-92-1 |
| Sample type |
SRA |
| |
|
| Source name |
Hamster Lung Tissue
|
| Organism |
Mesocricetus auratus |
| Characteristics |
tissue: Lung
gender: Female
age: 8-9 weeks
viral titre: 2 x 10 4 PFU/ml.
|
| Treatment protocol |
Virus stock was diluted with Phosphoate-buffered saline (PBS) to 2 x 104 PFU/ml. Hamsters with anesthetized and then intranasally inoculation with 50 ul of diluted viruses containing 103 PFU of viruses. Nasal washes were collected from hamster at 3 dpi and 5 dpi.
|
| Growth protocol |
Viruses were isolated from 5 confirmed COVID-19 patients for this study. For animal challenge, viral stocks were prepared after two serial passages in Vero E6 cells (ATCC; CRL-15786) in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) supplemented with 5% Fetal Bovine Serum (Thermo Fisher Scientific), and 100 IU penicillin G/ml and 100 ml streptomycin sulfate/ml, (Thermo Fisher Scientific).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Lung left inferior lobe from hamsters were cut into pieces and lysed with RA1 lysis buffer provided with the NucleoSpin® RNA Plus kit (Macherey-nagel), RNA extraction was performed according the manufacture’s instruction, including an on-column genomic DNA digestion step.
1 µg of high-quality total RNA (RIN>8) was used as starting material for cDNA library preparation with KAPAmRNA HyperPrep Kit. In brief, Poly-A containing mRNA was collected by using poly-T oligo-attached magnetic beads. The purified mRNA was fragmented to 200 – 300 bp by incubating at 94oC for 6 min in the presence of magnesium ions. The fragmented mRNA was then applied as template to synthesize the first-strand cDNA by using random hexamer-primer and reverse transcriptase. In the second strand cDNA synthesis, the mRNA template was removed and a replacement strand was generated to form the blunt-end double-stranded (ds) cDNA. The ds cDNA underwent, 3’ adenylation and indexed adaptor ligation. The adaptor-ligated libraries were enriched by 10 cycles of polymerase chain reaction (PCR). The libraries were denatured and diluted to optimal concentration.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Using software from Illumina (bcl2fastq), sequencing reads were assigned into individual samples with each sample having an average throughput of 87.7Gb and a total throughput of 11.7Gb. In terms of sequence quality, an average of 94% of the bases achieved a quality score of Q30 where Q30 denotes the accuracy of a base call to be 99.9%.
Fastq reads were subsequently aligned to the Golden Hamster and SARS-CoV-2 genome (NCBI Reference Sequence: NC_045512.2 ) with STAR (V2.7.2a) and counts were generated through its in-built htseq-count algorithm. (STAR parameters used: --readFilesCommand gunzip -c --outFilterScoreMinOverLread 0.3 --outFilterMatchNminOverLread 0.3 --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts).
Genes with a total read count below 10 were excluded from downstream analysis.
Differential Expression Analysis utilised DESeq2 which also was used to normalised expression values.
All analysis was performed through the R environment.
Genome_build: MesAur1.0
Supplementary_files_format_and_content: Raw counts and normalized expression tables are in comma-separated (.csv) format with genes distributed as rows and samples as columns.
|
| |
|
| Submission date |
Aug 10, 2020 |
| Last update date |
Sep 25, 2020 |
| Contact name |
Honglin Chen |
| E-mail(s) |
hlchen@hku.hk
|
| Organization name |
The University of Hong Kong
|
| Department |
Department of Microbiology
|
| Lab |
State Key Laboratory for Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine
|
| Street address |
Rm 5-19, 5/F, Lab block, 21 Sassoon Road, Pokfulam
|
| City |
Hong Kong |
| ZIP/Postal code |
0000 |
| Country |
Hong Kong |
| |
|
| Platform ID |
GPL28997 |
| Series (1) |
| GSE156005 |
SARS-CoV-2 infection induces transcriptomic alterations specific to activation of innate immunity and cellular distress. |
|
| Relations |
| BioSample |
SAMN15776922 |
| SRA |
SRX8920669 |
