10,000 red pulp macrophages (lineage-, autofluorescent, F4/80+, B220-, CD11bint, CD11cint) were flow-sorted directly into 1.5 ml eppendorf tubes containing 1 ml Trizol Reagent (ThermoFisher, #15596026). Tubes were inverted ten times and incubated for 5 minutes at room temperature before snap freezing and storage at - 80°C.
Growth protocol
C57Bl/6J mice were bred and housed in individually ventilated cages under specific pathogen free conditions (22°C, 50% humidity) at the University of Edinburgh, UK. Malaria infections were initiated when mice were approximately 9 weeks of age by intravenous injection of 200 sporozoites (according to our published protocol: Spence, P.J., et al., Mosquito transmission of the rodent malaria parasite Plasmodium chabaudi. Malar J, 2012. 11: p. 407). We mosquito transmitted two distinct Plasmodium chabaudi chabaudi clones, which we obtained from the European Malaria Reagent Repository (malariaresearch.eu) at the University of Edinburgh: P. chabaudi AS (28AS11) and P. chabaudi AJ (96AJ15). Anopheles stephensi mosquitoes (strain SD500) were reared in house at the University of Edinburgh. we initiated all experimental infections with sporozoites, since we have previously shown that mosquito transmission resets expression of the large sub-telomeric multi-gene families that control parasite virulence, and in turn shapes the host immune response during the pathogenic blood-stage of malaria infection (Spence, P.J., et al., Vector transmission regulates immune control of Plasmodium virulence. Nature, 2013. 498(7453): p. 228-31). 100 days after infection parasitaemia resolves without intervention.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using 1 - Bromo 3 - chloropropane (Sigma, #B9673) and Isopropanol (VWR, #437423R).
Label
biotin
Label protocol
RNA was processed according to manufacturers instruction using the GeneChiP Whole Transcript Pico Reagents Kit and run on the mouse exon 1.0 ST array (both Affymetrix) by Hologic (Manchester, UK).
Hybridization protocol
According to manufacturers instruction GeneChiP Whole Transcript Pico Reagents Kit.
Scan protocol
According to manufacturers instruction Affymetrix mouse exon 1.0 ST array
Data processing
Data were processed in R/Bioconductor using the oligo, pd.mta.1.0 and mta10sttranscriptcluster packages. Transcript level analyses were performed using limma and eBayes on rma() normalised data.
Tissue printing: splenic red pulp macrophages of once-malaria infected mice are transcriptionally identical to prenatally seeded red pulp macrophages from uninfected mice.
