Comment[ArrayExpressAccession] E-GEOD-41355 MAGE-TAB Version 1.1 Public Release Date 2013-04-23 Investigation Title IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling Comment[Submitted Name] IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling Experiment Description Myeloid dendritic cells from WT, Irf3-/-xIrf7-/-, Irf3-/-xIrf5-/-xIrf7-/-, Mavs-/- (IPS1-/-)and Ifnar-/- mice were infected with West Nile virus to assess the contributions of specific signaling and transcription factors in initiating the antiviral response RPMI + rmGM-CSF purified bone marrow derived myeloid DC (mDC), mock and WNV infected with insect derived virus and RNA isolated at 24 hours post infection . Each genotype has matched mock and infected samples. n=6 for WT mock, WT infected, IFNAR mock, IFNAR infected, IRF3x7 infected; n=5 IRF3x7 infected; n=3 IRF3x5x7 mock, IRF3x5x7 infected, MAVS mock, MAVS infected. RNA was prepared using the Illumina bead station assay and hybridized to Illumina RefSeq-8 V2 BeadChips. Note: MAVS=IPS1 Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Wilkinson Lazear Lancaster Wilkins Suthar Huang Vick Clepper Thackray Brassil Virgin Nikolich-Zugich Moses GaleJr Fruh Diamond Person First Name Peter Helen Alissa Courtney Mehul Albert Sarah Lisa Larissa Margaret Herbert Janko Ashlee Michael Klaus Michael Person Mid Initials A M S C M W V S Person Email geo@ncbi.nlm.nih.gov Person Affiliation Vaccine and Gene Therapy Institute Person Phone 772-345-5711 Person Address Bioinformatics, Vaccine and Gene Therapy Institute, 9801 Discovery Way, Port Saint Lucie, Florida, USA Person Roles submitter Protocol Name P-GSE41355-1 P-GSE41355-5 P-GSE41355-6 P-GSE41355-2 P-GSE41355-3 P-GSE41355-4 P-GSE41355-7 Protocol Description 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 ID_REF = VALUE = Quantile normalized, log2 transformed intensities The hybridized BeadChips were washed, blocked, stained and scanned according to the Illumina's direct 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 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. 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. 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. The arrays were scanned using an Illumina iScan scanner and quantified using GenomeStudio software (Illumina). Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name SUBJECT GENOTYPE INFECTION Experimental Factor Type subject genotype infection Publication Title IRF-3, IRF-5, and IRF-7 coordinately regulate the type I IFN response in myeloid dendritic cells downstream of MAVS signaling. Publication Author List Lazear HM, Lancaster A, Wilkins C, Suthar MS, Huang A, Vick SC, Clepper L, Thackray L, Brassil MM, Virgin HW, Nikolich-Zugich J, Moses AV, Gale M Jr, Fr�h K, Diamond MS PubMed ID 23300459 Publication DOI 10.1371/journal.ppat.1003118 Comment[SecondaryAccession] GSE41355 Comment[GEOReleaseDate] 2013-04-23 Comment[ArrayExpressSubmissionDate] 2012-10-04 Comment[GEOLastUpdateDate] 2013-04-24 Comment[AEExperimentType] transcription profiling by array Comment[AdditionalFile:Data1] GSE41355_non-normalized.txt SDRF File E-GEOD-41355.sdrf.txt