Transcriptomics of pathogen Cronobacter turicensis z3032 in lysogenic broth
Cronobacter (C.) is an important emerging opportunistic foodborne pathogen representing  significant cause of mortality in neonatal patients with bacteremia and meningitis.  Knowledge on the pathobiology of Cronobacter mediated meningitis has to a large extend been explored using in vitro models. To explore the innate immune response against the neonatal sepsis/meningitis causing isolate C. turicensis z3032 in vivo, zebrafish larvae (Danio rerio) were used as infection model. Following establishment of infection in zebrafish larvae with z3032, dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the head region of the zebrafish host. Overall design: returned to a petri dish with fresh embryo medium and incubated at 28 °C until development of typical signs of disease such as axis curvature and necrosis which was observed at approx. 36 h dpi. 60 randomly chosen zebrafish larvae from each group (infected, mock-infected) were euthanized by a prolonged immersion in an overdose of 50 mg/L Tricaine solution, followed by decapitation with a sharp blade, their heads collected and transferred into 1.5ml Eppendorf tubes Excess E3 medium was removed and  replaced by RNAlater (Ambion) and stored at 4°C until further processing. For the extraction of total RNA, RNAlater was replaced by 600 µl of Qiazol, and the tissue was homogenized using a pestle motor followed by drawing of sample with a needle until the tissue was completely homogenized. As a control, RNA was isolated from z3032 cultures grown in LB to stationary phase (18 – 24 h). For both the zebrafish and bacterial culture, RNA extractions were performed using the miRNeasy Mini kit (Qiagen) according to the manufacturer's protocols. An additional DNase treatment was performed with a TURBO DNA-free kit (Ambion) according to the manufacturer's protocols. The concentration of the extracted RNA was measured using a NanoDrop Spectrophotometer. The total RNA samples were subjected to ribosomal RNA (rRNA) depletion, zebrafish samples mock-infected with PBS were treated with Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) (Illumina), zebrafish infected with C. turicensis were treated with Ribo-Zero rRNA Removal Kit (Gram-negative Bacteria) (Illumina) followed by Ribo-Zero Gold rRNA Removal Kit (Human/Mouse/Rat) and the C. turicensis sample grown in LB was treated with Ribo-Zero rRNA Removal Kit (Gram-negative Bacteria). The depletion steps and subsequent 125 bp paired-end RNA Sequencing on a HiSeq2500 (Illumina) were performed at the Functional Genomic Center at University Zurich (FGCZ), Zurich, Switzerland.