gender: Female strain: B6.GzmB-/- age: 8-10 weeks treatment: Plasmodium berghei ANKA time point: Day 4 post-infection tissue: Spleen cell type: CD4 T cells
Treatment protocol
Mice were infected intravenously (i.v.) with 100,000 PbA-parasitized red blood cells (pRBCs). At day 4 post-infection, animals were sacrificed by CO2 asphyxiation, in accordance with animal ethics guidelines. Following sacrifice, spleens were removed. CD4+ T cells were isolated by fluorescence activated cell sorting. Cells were homogenised using QIAshredder spin columns and stored in RLT buffer from the RNeasy mini kit (Qiagen, Doncaster, Australia).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy mini kit, according to manufacturer’s instructions, and contaminants were removed by passing samples over RNeasy mini-columns with on-column DNase treatment (Qiagen). RNA integrity was assessed by denaturing agarose gel electrophoresis, samples were included based on 28S:18S ratios ≥ 1.7. RNA concentrations were assessed using the Nanodrop ND-1000 (NanoDrop Technologies Inc, Wilmington, DE).
Label
Cy3
Label protocol
Biotinylated cRNA was prepared with the Illumina TotalPrep RNA Amplification Kit (Ambion, Austin, TX).
Hybridization protocol
Labelled cRNA was hybridized to Sentrix BeadChip Arrays, MouseWG-6 v2 (Illumina Inc, San Diego, CA, USA) according to standard Illumina protocols.
Scan protocol
Standard Illumina scanning protocol.
Data processing
The data were extracted in GenomeStudio (Illumina) using default analysis settings and no normalization method. The resulting data were imported into GeneSpring GX v11.0 (Agilent Technologies). The expression values were normalized using Percentile Shift Normalization with default settings. Briefly, Percentile Shift Normalization is a global normalization, where the location of all the spot intensities in an array are adjusted. This normalization takes each column in an experiment independently, and computes the nth percentile of the expression values for this array, across all spots (where n has a range from 0-100 and n=50 is the median; n = 75 for this experiment). It then subtracts this value from the expression value of each entity. In GeneSpring GX, log transformation is done on the dataset before the normalization and hence the percentile is subtracted from the expression value.
