2 protocols
normalization data transformation protocol
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.
nucleic acid library construction protocol
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).