RNA-seq of mouse splenocytes following infection with Listeria monocytogenes

C57BL/6 mice were maintained in specific pathogen-free conditions in accordance with the Institutional Animal Care and Use Committee. Listeria monocytogenes (clinical isolate 10403s) was from V. Boyartchuk (NTNU, Trondheim, Norway) and infected as previously described (PMID 24477914) for 24 hours before harvesting the spleen. PBS was used as control. . Single cell suspensions from spleens were used to make total RNA. 4 µg of total RNA was used to generate libraries for RNA-sequencing (Illumina unstranded mRNA kits). Samples were sized, quantified and validated on a Bioanalyzer. Libraries were sequenced on a High-Seq System (Illumina 2000, San Diego, CA) as paired-end 100 reads. Sequence reads were aligned to the mouse genome (assembly NCBI m37/mm9) using TopHat.Gene level read counts based on the resulting alignments were calculated using HTSeq. The DESeq R package was used to normalize gene counts, calculate fold change in gene expression, estimate p-values and adjusted p-values for change in gene expression values, and to perform a variance stabilized transformation on read counts.