title: RNAseq of Coxiella burnetii 1 day post infection of Galleria mellonella larvae; replicate 3
design: Galleria mellonella larvae were infected with 10^6 GE of Coxiella burnetii NMII; haemocytes were collected at various time points by centrifugation, re-suspended in 1 ml TRIzol Reagent and frozen at -80C. Samples stored frozen in TRIzol Reagent were thawed and total RNA was extracted using the Direct-zol RNA MiniPrep Kit. Contaminating DNA was digested by DNAse I treatment 5 g RNA isolated from infected larvae, and 2 g RNA isolated from bacterial culture (both in triplicate) was further processed: eukaryotic mRNA was depleted from the samples extracted from infected G. mellonella using TruSeq mRNA stranded (oligo-dT) beads (TruSeq RNA Library Preparation Kit v2, Illumina RS-122-2001). The depleted RNA (eukaryotic rRNA and total bacterial RNA) as well as the total C. burnetii RNA isolated from bacterial culture (bacterial rRNA and bacterial mRNA) was then enriched for bacterial mRNA using the riboPOOL Human:Pan-prokaryote or the riboPOOL Pan-prokaryote kits, respectively (Source Bioscience rdS0501 or rdCS0501, respectively). RNA-seq libraries were prepared using the Illumina TruSeq Stranded mRNA Library Prep Kit according to the manufacturers protocol.. Sequencing was performed using an Illumina HiSeq 2500 sequencing instrument at 125-bp read length, paired end.