|
| Status |
Public on Apr 23, 2013 |
| Title |
IRF3/5/7 TKO 4b |
| Sample type |
RNA |
| |
|
| Source name |
RPMI + rmGM-CSF purified BM-DC populations, mock and WNV infected with insect derived virus and RNA isolated 24 hours post infection.
|
| Organism |
Mus musculus |
| Characteristics |
subject: Mouse_14
genotype: IRF3/5/7 TKO
cell type: bone marrow derived myeloid DC bmDCs
infection: West Nile Virus
time point: 24 hours
|
| Treatment protocol |
GM-CSF was replenished after 2 days and non-adherent cells were sub-cultured after 4 days. Sub-cultured cells were infected at an MOI of 25 with WNV-NY. To determine the percentage of cells infected, mDC were detached 24 h after WNV inoculation. Non-specific binding of antibody to Fc-γR was blocked with anti-CD16/CD32 (Biolegend), and all subsequent surface staining (I-Ab-FITC, CD86-PE, and CD11c-APC (all from Biolegend)) was performed in PBS supplemented with 2% fetal calf serum. Cells were fixed, permeabilized, and then stained with Alexa-647 conjugated anti-WNV E16 anti-WNV MAb. For microarray analysis, total RNA was harvested at 24 hours post-infection.
|
| Growth protocol |
Bone marrow was cultured in RPMI supplemented with 10% fetal bovine serum, penicillin/streptomycin, L-glutamine, non-essential amino acids, 55 μM β-mercaptoethanol and 20 ng/ml recombinant mouse GM-CSF (eBioscience) for 6 days in non-tissue culture treated plates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The DC/Mac populations were harvested at each desired post-infection time point. The cells were washed once with RT 1XPBS (about 2ml/Well). Then 0.5ml added of warmed trypsin to each well, incubate 10 min at 37°C. Once this was done, 5 ml added of cold PBS w/ 10% serum or use 10% complete medium. The cells were then collected in 15ml conical tube. Cells were spun down at 1000-1200 RPM x 5 minutes at 4°C.
|
| Label |
Biotin
|
| Label protocol |
The hybridized BeadChips were washed, blocked, stained and scanned according to the Illumina's direct hybridization protocol.
|
| |
|
| Hybridization protocol |
Microarray analysis was conducted using 750ng of biotinylated cRNA hybridized to Human RefSeq-8 V2 BeadChips (Illumina) at 58°C for 20 hours, according to Illumina's direct hybridization protocol
|
| Scan protocol |
The arrays were scanned using an Illumina iScan scanner and quantified using GenomeStudio software (Illumina).
|
| Description |
bmDC from Mouse_14, wnv infected
|
| Data processing |
Analysis of the GenomeStudio output data was conducted using the R statistical language and various software packages from Bioconductor. Missing values were imputed using the KNN algorithm from the impute R package. Quantile normalization was applied, followed by a log2 transformation
|
| |
|
| Submission date |
Oct 04, 2012 |
| Last update date |
Apr 23, 2013 |
| Contact name |
Peter A Wilkinson |
| Organization name |
Case Western Reserve University
|
| Department |
Pathology / Systems Biology & Bioinformatics
|
| Street address |
2103 Cornell Road
|
| City |
Cleveland |
| State/province |
Ohio |
| ZIP/Postal code |
44120 |
| Country |
USA |
| |
|
| Platform ID |
GPL6885 |
| Series (1) |
| GSE41355 |
IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling |
|
