|
| Status |
Public on Apr 04, 2015 |
| Title |
WCN003_WNVE218A_12h_3_mRNA |
| Sample type |
RNA |
| |
|
| Source name |
Primary cortical neurons, inoculated with WNVE218A, 12 hour(s), bioreplicate 2, technical replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6J
embryonic stage: E15
developmental stage: embryo
tissue: brain
time: 12
virus: WNVE218A
technical_replicate: 3
biological_replicate: 2
|
| Treatment protocol |
On day 4, triplicate technical replicates of cortical neurons were mock-inoculated or inoculated with plasmid-derived wild type West Nile virus NY99 clone 382 (WNVWT) or a plasmid-derived isogenic E218A mutant West Nile virus NY99 clone 382 (WNVE218A). After 1 hr of infection at 37oC, virus inoculum was removed. Neurons were then cultured for 0-23 additional hours at 37oC. At 1, 8, 12 and 24 hours after inoculation medium was removed and RNA harvested from adherent neurons using Trizol.
|
| Growth protocol |
Mouse fetal brain was taken from C57Bl/6J mouse embryos at E15. After removing meninges, cortical tissue was dissociated, filtered and plated in neurobasal medium (with B27 supplement, Glutamax and penicillin/streptomycin) at a density of 5 x 105 cells/well in a poly-D-lysine and laminin coated 24-well dish. Three days after plating (D2), cell medium was replaced with fresh medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from Trizol.
|
| 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 × Blocking Agent and 2.2 μl of 25×Fragmentation Buffer, then heated at 60 °C for 30 min, and finally 55 μl 2 × 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 with using the Agilent DNA Microarray Scanner (part number G2505C).
|
| 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 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 two biological replicates was performed independently of the other.
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| |
|
| Submission date |
Mar 31, 2015 |
| Last update date |
Aug 31, 2016 |
| 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 |
GPL11202 |
| Series (2) |
| GSE65575 |
Modeling Host Responses to Understand Severe Human Virus Infections |
| GSE67473 |
Mouse cortical neuron transcriptional response to wild-type West Nile virus (WNV-NY) and mutant virus WNV E218A [mRNA] |
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