|
| Status |
Public on Apr 29, 2015 |
| Title |
WGCN003_mock_12h_2_mRNA |
| Sample type |
RNA |
| |
|
| Source name |
Primary granule cell neurons, mock-inoculated, 12 hour(s), bioreplicate 2, technical replicate 2
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6J
age: day 6
developmental stage: neonatal
time: 12
virus: mockulum
technical_replicate: 2
biological_replicate: 2
|
| Treatment protocol |
On day 4, triplicate technical replicates of granule cell 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 37C, virus inoculum was removed. Neurons were then cultured for 0-23 additional hours at 37C. At 1, 8, 12 and 24 hours after inoculation medium was removed and RNA harvested from adherent neurons using Trizol.
|
| Growth protocol |
Mouse neonatal brain was taken from day 6 C57Bl/6J mouse pups. After removing meninges and choroid plexus, cerebellar tissue was dissociated, filtered and plated in neurobasal medium (with B27 supplement, Glutamax and penicillin/streptomycin) at a density of 5 x 10^5 cells/well in a poly-D-lysine coated 24-well dish. Two days after plating (D1), 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 |
Apr 28, 2015 |
| Last update date |
Aug 31, 2016 |
| Contact name |
Natalie Heller |
| E-mail(s) |
[email protected]
|
| 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 |
| GSE68380 |
Mouse granule cell neuron transcriptional response to wild-type West Nile virus (WNV-NY) and mutant virus WNV E218A (mRNA) |
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