Virus and mice
Original stocks of mouse adapted A/Puerto Rico/8/34 (H1N1, PR8M) virus were obtained from Stefan Ludwig, University of Münster (Blazejewska et al., 2011). Virus stocks were propagated in the chorioallantoic cavity of ten-day-old pathogen-free embryonated chicken eggs for 48 h at 37 °C as described previously (Wilk and Schughart, 2012). Viral titer was determined by focus-forming unit (FFU) assay as described previously (Wilk and Schughart, 2012).
Infection of mice. Female mice at the age of 8-12 weeks were anesthetized by intra-peritoneal injection of Ketamine/Xylazine (10%(v/v) of 100 mg/ml ketamine and 5%(v/v) of 20 mg/ml Xylazine in 0.9% (w/v) NaCl) with a dose adjusted to the individual body weight (200 µl/20 g body weight). Infection was performed by intranasal application of virus solution in 20 µl sterile phosphate-buffered saline (PBS).
RNA isolation and sequencing. Mice were sacrificed at day 3 post infection and entire lungs were extracted. The lungs were immediately transferred to RNAlater solution (Qiagen), kept at 4 °C for one day and subsequently stored at -20 °C. RNA was isolated using Qiagen Midi Kit as described previously (Alberts et al., 2010). RNA quality was controlled on a 2100 Bioanalyzer Instrument (Agilent).500ng of total RNA was used to prepare libraries for sequencing using the Lexogen SENSE mRNA-seq library kit for Ion Torrent. Libraries were amplified for 11 cycles as the final step of library preparation.
Before sequencing, 1ul aliquots of this material was pooled and sequenced on an Ion Torrent PGM 314 chip. Barcode quantification data from the PGM were used to balance the barcodes for final pooling before sequencing. Following this final pooling, the library pools were sized to a target size of 260bp on a Pippin Prep instrument using the 2% Pippin Agarose gel. The sized libraries were examined on an Agilent High Sensitivity DNA chip, quantified using real-time PCR, and used to prepare spheres using a One-Touch 2 device. These spheres were then sequenced on an Ion Torrent Proton sequencer with a P1 chip. On average, 67 million reads were obtained per sample.
Bioinformatic analyses. RNAseq reads were quality-trimmed using Trimgalore and then mapped to the mouse mm10 reference genome or the influenza A virus PR8M genome using STAR. Read counts were summarized for genes as features using the R-package Rsubread, normalized and log2 transformed using the R-package DESeq2 and subsequently batch-corrected using the combat function of the R-package sva.
For annotations of genes, ENTREZ-ID from Rsubread were matched to RefSeq annotations using R-package biomart. Duplicate RefSeq annotations were given the extension _1, _2, etc.
Alberts, R., Srivastava, B., Wu, H., Viegas, N., Geffers, R., Klawonn, F., Novoselova, N., do Valle, T.Z., Panthier, J.J., and Schughart, K. (2010). Gene expression changes in the host response between resistant and susceptible inbred mouse strains after influenza A infection. Microbes Infect 12, 309-318.
Blazejewska, P., Koscinski, L., Viegas, N., Anhlan, D., Ludwig, S., and Schughart, K. (2011). Pathogenicity of different PR8 influenza A virus variants in mice is determined by both viral and host factors. Virology 412, 36-45.
Wilk, E., and Schughart, K. (2012). The mouse as model system to study host-pathogen interactions in influenza A infections. Curr Protoc Mouse Biol 2, 177-205.
QC analysis of the RNAseq results from the flu-infected BXD mice (one mouse per strain)
- Total of 513 genes have LRS above 23 (LOD=5) and 65 have LRS above 46 (LOD=10). Highest LRS of 125.6. N of 43 strains with no replicates and no pooling. Top record is Rufy4 (high in D).