Computational protocol: Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19

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Protocol publication

[…] Frozen BMDMs were thawed and plated with supplemented DMEM as described above. On day 12, cells were counted and plated onto a 12-well plate at a density of 3 × 106 per well. The next day, cells were washed and bacteria (LVS) were added at an MOI of 100. Total RNA was extracted (RNeasy; Qiagen) at time zero (no bacteria) and at 2, 4, and 10 h after infection and was immediately stored at −80°C. Illumina library preparation, sequencing, and data processing were done as previously described (). In brief, mRNA was purified from total RNA by poly(A)-tail enrichment (Dynabeads mRNA purification kit; Invitrogen), fragmented into 200 to 400 bp, and reverse transcribed into cDNA before Illumina sequencing adapters were added to each end. Libraries were barcoded, multiplexed into four samples per sequencing lane in the Illumina HiSeq 2000 system, and sequenced from both ends; this process resulted in 40-bp reads after the bar codes were discarded.The RNA-seq reads were mapped to a synthetic mouse genome (mm10) in which all the single polymorphic nucleotides (as annotated by Wellcome Trust Sanger Institute sequencing; between AJ and B6 were masked with Bowtie2 (2.2.3) () and TopHat (v2.0.4) (). Gene expression levels were then estimated as fragments per kilobase of exon per million fragments mapped (FPKM) in CuffLinks (v2.2.0) (). At each time point, gene fold changes from baseline (defined as RNA levels from resting, uninfected, and unstimulated cells) were determined for both AJ and B6 BMDMs. The ranked lists of the fold changes (.rnk files) were analyzed by GSEA to determine which cellular pathways were upregulated, downregulated, or unchanged. With this platform, a total of 917 pathways were analyzed and scored. For samples from 2 and 10 h after infection, specific genes from each of the top 10 upregulated immunological pathways were combined and analyzed. Sorting and visualization of the data was done using SPIN (). Genes with a 1.5-fold or greater difference between the AJ and B6 results were plotted. […]

Pipeline specifications

Software tools Bowtie2, TopHat, Cufflinks
Application RNA-seq analysis
Organisms Francisella tularensis, Mus musculus, Bacteria, Homo sapiens
Diseases Communicable Diseases, Infection