|
| Status |
Public on May 06, 2016 |
| Title |
EU937001_wild_8h_3_microRNA |
| Sample type |
RNA |
| |
|
| Source name |
U937-VP30 cells, wild-inoculated, 8h
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U937-VP30
cell type: U937 monocyte-like cells expressing the Ebola VP30 protein
treatment: Zaire Ebola virus wild-type in the delta-VP30 background
time (post-infection): 8h
biological replicate: 3
|
| Treatment protocol |
Cells were infected with a multiplicity of infection of 1.0.
|
| Growth protocol |
U937-VP30 cells are grown on 10% fetal bovine serum (FBS) in RPMI 1640 medium. Cells were at passage 8 when infected. Virus growth medium after infection was the same; 10% FBS-RPMI 1640.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Extract was 1mL of Trizol per well.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was labeled according to Agilent's Quick Amp Labeling protocol as part of the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technology). Total RNA from each sample was linearly amplified and labeled with Cy3-UTP.
|
| |
|
| Hybridization protocol |
Hybridization was performed according to Agilent One-Color Microarray-Based Gene Expression Analysis Protocol (Agilent Technology). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000. 1 μg of each labeled cRNA was fragmented by adding 11 μl 10 x Blocking Agent and 2.2 μl of 25xFragmentation Buffer, then heated at 60 °C for 30 min, and finally 55 μl 2 x GE Hybridization buffer was added to dilute the labeled cRNA. 100μl of hybridization solution was dispensed into the gasket slide and assembled to the gene expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
|
| Scan protocol |
The hybridized arrays were washed, fixed and scanned using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Description |
microRNA
|
| Data processing |
Scanned images were analyzed using Agilent Feature Extraction Software (v11.0.1.1). The limma package for R (available on Bioconductor) was used to perform background correction, quantile normalization (normalizeBetweenArrays), and summarization (avereps) to derive a single normalized intensity value (log2) per probe. Outlier samples were detected using PCA and by visual inspection of heatmaps, and all data was re-processed after removing outlier samples. All data processing for each of the biological replicates was performed independently of the other.
|
| |
|
| Submission date |
Apr 29, 2016 |
| Last update date |
May 20, 2018 |
| Contact name |
Natalie Heller |
| E-mail(s) |
natalie.heller@pnnl.gov
|
| Organization name |
PNNL
|
| Street address |
902 Battelle Blvd.
|
| City |
Richland |
| ZIP/Postal code |
99354 |
| Country |
USA |
| |
|
| Platform ID |
GPL19730 |
| Series (2) |
| GSE65575 |
Modeling Host Responses to Understand Severe Human Virus Infections |
| GSE80833 |
Human U937 cell transcriptome response to Zaire Ebola virus wild-type in the ΔVP30 background and Δmucin virus [miRNA] |
|
