Isolate rna mini kit

To measure transcriptional levels for either RT-PCR or in Hiseq RNA transcriptome analysis conducted by SA Pathology (Central Adelaide local Health Network via an Illumina platform), cells were isolated and RNA extracted as below. Bacterial cells were harvested from either semi-solid media (0.25 % LB agar) for RT-PCR using pre-chilled PBS or harvested from Mueller Hinton broth at OD₆₀₀ of 0.6 for Hiseq. Bacterial cells were pelleted by centrifugation at 4500 g for 15 min and lysed in 1 mL TRIzol reagent (Life Technologies, Australia) and 200 μL of chloroform. Following phase separation, the aqueous layer was collected and RNA isolated using the Isolate RNA Mini Kit (Bioline, NSW, Australia) following the manufacturer’s recommendations. Samples for RT-PCR were subsequently treated with DNaseI (Promega, WI, USA) for 30 min at 37 °C and 1 μL of stop solution was added before a final incubation for 10 min at 65 °C. Triplicate samples were pooled and sent to SA Pathology for Ribosomal RNA reduction using Epicentre Ribo-Zero kit and RNA library bar-coding was undertaken on RNA for HiSeq. Samples were run on the Illumina HiSeq platform (1X50 base pair single reads). These data are accessible through GEO Series accession number GSE64935 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE64935).

RNA was extracted with the ISOLATE RNA Mini Kit (Bioline) and quantified by real‐time PCR using SensiFAST™ Probe Hi‐ROX One‐Step Kit (Bioline) allowing reverse transcription and PCR amplification in a single step. The real‐time PCR was performed in a Roche LightCycler 96. The Renilla luciferase, β‐actin, and pBAC‐SARS‐CoV replicon qPCR primers are listed in Appendix Table S2. Experiments were performed in at least triplicates.