Comment[ArrayExpressAccession] E-GEOD-49932 MAGE-TAB Version 1.1 Public Release Date 2014-03-03 Investigation Title RNA-Seq global gene expression of four Dendritic cells subpopulations from lung (CD8+ and CD8- pDCs, CD103-CD11b+ and CD103+CD11b- cDCs) of influenza infected mice at four time points following infection Comment[Submitted Name] RNA-Seq global gene expression of four Dendritic cells subpopulations from lung (CD8+ and CD8- pDCs, CD103-CD11b+ and CD103+CD11b- cDCs) of influenza infected mice at four time points following infection Experiment Description Hundreds of immune cell types work in coordination to maintain tissue homeostasis. Upon infection, dramatic changes occur with the localization, migration and proliferation of the immune cells to first alert the body of the danger, confine it to limit spreading, and finally extinguish the threat and bring the tissue back to homeostasis. Since current technologies can follow the dynamics of only a limited number of cell types, we have yet to grasp the full complexity of global in vivo cell dynamics in normal developmental processes and disease. Here we devise a computational method, digital cell quantification (DCQ), which combines genomewide gene expression data with an immune cell compendium to infer in vivo dynamical changes in the quantities of 213 immune cell subpopulations. DCQ was applied to study global immune cell dynamics in mice lungs at ten time points during a 7-day time course of flu infection. We find dramatic changes in quantities of 70 immune cell types, including various innate, adaptive and progenitor immune cells. We focus on the previously unreported dynamics of four immune dendritic cell subtypes, and suggest a specific role for CD103+CD11b- cDCs in early stages of disease and CD8+ pDC in late stages of flu infection. To better understand the physiological role of these differential dynamic changes in the DCs, we measured the genome-wide RNA expression of all four DC subpopulations from lung of influenza infected mice at four time points following infections (two mice per time-point). For sorting dendritic cells from lungs, the lungs from infected and control uninfected C57BL/6J mice were immersed in cold PBS, cut into small pieces in 5 ml DMEM media containing 10% Bovine Fetal Serum, the cell suspensions were grinded using 1ml syringe cup on a 70 μm cell strainers (BD Falcon). The cells were washed with ice cold PBS. Remaining red blood cells were lysed using ammonium chloride solution (Sigma). Cells were harvested, immersed 1ml FACS buffer [PBS+2% FBS, 1mM EDTA], Fc receptors were blocked with anti-mouse CD16/CD32, washed with FACS buffer and divided into two tubes for sorting cDC and pDC cells. 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 Gat-Viks Michal Irit Ido Person First Name Irit Mandelboim Gat-Viks Amit Person Email geo@ncbi.nlm.nih.gov Person Affiliation Tel-Aviv University Person Address Department of Cell Research and Immunology, Tel-Aviv University, Tel-Aviv, Tel-Aviv, Israel Person Roles submitter Protocol Name P-GSE49932-2 P-GSE49932-1 Protocol Description All reads were aligned to the mouse reference genome (NCBI 37, MM9) using the TopHat aligner The raw expression levels of the genes were calculated using Scripture, an ab-initio software which reconstruct transcriptomes. Normalization was done using DESeq based on the negative binomial distribution and a local regression model Genome_build: mm9 Supplementary_files_format_and_content: tab-delimited text file which includes RefSeq ID and normalized read counts for each sample. For sorting dendritic cells from lungs, the lungs from infected and control uninfected C57BL/6J mice were immersed in cold PBS, cut into small pieces in 5 ml DMEM media containing 10% Bovine Fetal Serum, the cell suspensions were grinded using 1ml syringe cup on a 70 μm cell strainers (BD Falcon). The cells were washed with ice cold PBS. Remaining red blood cells were lysed using ammonium chloride solution (Sigma). Cells were harvested, immersed 1ml FACS buffer [PBS+2% FBS, 1mM EDTA], Fc receptors were blocked with anti-mouse CD16/CD32, washed with FACS buffer and divided into two tubes for sorting cDC and pDC cells. For sorting cDC, the cells were stained with antibodies against multiple surface antigens: Percp cy5.5 conjugated anti CD45 (clone –F11), APC conjugated CD11c (clone N418), PB conjugated anti I-A/IE (clone M5/114.15.2), PE conjugated anti CD103 (clone 2E7) and FITC conjugated CD11b (clone M1/70). The cDC cells were identified as CD45 positive, CD11c high, MHC-II positive, and were gated as CD103 negative CD11b positive and CD103 positive CD11b negative. For pDC sorting, the cells were stained with the following antibodies: Percp cy5.5 conjugated anti CD45 (clone –F11), APC conjugated CD11C (clone N418), APC CY7 conjugated CD45R/B220, (clone RA3-6B2), PE conjugated anti PDCA-1 (clone129c1) and PE CY7 conjugated CD8 (clone 53-6.7). The pDC cells were identified as CD45 positive, CD11C intermediate, B220 positive, PDCA-1 positive, and gated as CD8 positive and CD8 negative. Flow cytometry was performed using SORP FACSAriaII Flow Cytometer (Becton Dickinson) and data were analyzed using WinMDI 2.8 software. For preparation of RNA-seq libraries, total RNA was fragmented into average size of 300 nucleotides by chemical heat (95Oc) treatment for 4:30 minutes (NEBNext Magnesium RNA Fragmentation Module). The 3’ polyadenylated fragments were enriched by selection on poly dT beads (Dynabeads Invitrogen). Strand specific cDNA was synthesized using a poly T-VN oligo (18 T) and Affinity Script RT enzyme (Agilent). Double strand DNA was obtained using Second strand synthesis kit (NEB). DNA ends were repaired using T4 polynucleotide kinase and T4 polymerase (NEB-Next). After addition of an adenine base residue to the 5’ end using Klenow enzyme (NEB-Next), a barcode Illumina compatible adaptor (IDT) was ligated to each fragment. The washed DNA fragment was amplified by PCR (12 cycles) using specific primers (IDT) to the ligated adaptors. The quality of the library was analyzed by Tapestation (Agilent). Protocol Type normalization data transformation protocol nucleic acid library construction protocol Experimental Factor Name TIME INFECTION Experimental Factor Type time infection Publication Title Digital cell quantification identifies global immune cell dynamics during influenza infection. Publication Author List Altboum Z, Steuerman Y, David E, Barnett-Itzhaki Z, Valadarsky L, Keren-Shaul H, Meningher T, Mendelson E, Mandelboim M, Gat-Viks I, Amit I PubMed ID 24586061 Publication DOI 10.1002/msb.134947 Comment[SecondaryAccession] GSE49932 Comment[GEOReleaseDate] 2014-03-03 Comment[ArrayExpressSubmissionDate] 2013-08-16 Comment[GEOLastUpdateDate] 2014-05-19 Comment[AEExperimentType] RNA-seq of coding RNA Comment[SecondaryAccession] SRP028867 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR953151-SRR953178 SDRF File E-GEOD-49932.sdrf.txt