|
| Status |
Public on May 20, 2019 |
| Title |
WNV 2 |
| Sample type |
SRA |
| |
|
| Source name |
Lung carcinoma cells_west nile virus
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
cell compartment: Extracellular vesicles
|
| Treatment protocol |
Cells were infected with Kunjin strain of West Nile virus at multiplicity of infection (MOI) = 1 by incubation of cells with 45 μl of inoculum per cm2 of growth area for 2h at 37oC. After inoculum was removed, the cells were washed twice with PBS, and fresh medium was added. Human interferon alpha 2a (Roche, USA) was added to cells at final concentration of 300u/ml for 24h. Infected and IFN-treated cells cells were maintained in exosomes-free F12K medium (Gibgo, USA) supplemented with 2% FBS.
|
| Growth protocol |
Human lung carcinoma cells (A549) [ATCC CCL-185] were cultured in F12K medium supplemented with 10% heat-inactivated FBS,100 units penicillin ml−1, 100 µg streptomycin ml−1, 0.29 mg l-glutamine ml−1 (Gibco) and 1 mM sodium pyruvate. Cells were maintained at 37 °C in CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
EVs were isolated from conditioned media using ExoQuick-TC (Systems Bioscience, Mountain View, CA) per manufacturer’s protocol. To inactivate and remove contaminating virus particles prior to exosome extraction, 1u/ml RNaseA (Sigma, USA) was added to the conditioned media followed by heating at 56°C for 1 hour and centrifugation for 15 min at 3000 x g, 4oC. RNA was then extracted from pelleted extracellular vesicles using mirVana miRNA isolation Kit (Ambion, USA).
cDNA libraries were prepared using a TruSeq RNA Library Prep Kit (v2) (Illumina Inc., USA)
|
| |
|
| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
A549_EV_counts.xls
|
| Data processing |
Reads were mapped to human genome assembly hg38 using STAR v2.5.2 with default configurations
Reads were counted using HTSeq v0.7.0.1 with default configurations in union mode and counting to “exon” feature. For counting the RNA transcripts and small noncoding RNAs from EVs, gene feature format (GFF) files were obtained from UCSC genome browser (assembly GRCh38/hg38, Dec. 2013). GFF files with coordinates of miRNAs were obtained from miRBase (release 21).
Genome_build: hg38
Supplementary_files_format_and_content: .xls file as produced from HTSeq outputs and contains row counts for miRNA and snoRNAs
|
| |
|
| Submission date |
May 03, 2018 |
| Last update date |
May 20, 2019 |
| Contact name |
Andrii Slonchak |
| Organization name |
The University Of Queensland
|
| Department |
School of Chemistry and Molecular Biosciences
|
| Lab |
RNA Virology Lab
|
| Street address |
Bldg 76 Cooper Rd
|
| City |
St Lucia |
| State/province |
QLD |
| ZIP/Postal code |
4072 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE114008 |
Small RNA profiling of extracellular vesicles secreted by A549 cells infected with West Nile virus and treated with interferon alpha |
|
| Relations |
| BioSample |
SAMN09058818 |
| SRA |
SRX4035656 |
