BioSample

Protocols: To determine differential expression of genes in response to ethylzingerone, B.vietnamiensis strain G4 was cultured in a liquid growth medium without antimicrobial (control condition) and with 0.5x the MIC (test condition). Four biological replicates (each with 4 technical replicates) were obtained for both conditions, and the test results compared to that of the control. After exposure to the control and test condition for 8.5 and 24 hours respectively, the culture samples were rapidly cooled using liquid nitrogen and cells pelleted by centrifugation at 20,000 xg at 4°C for 1 minute. The culture supernatant was removed, and the pelleted cells were snap-frozen and stored at -80°C. Total RNA was extracted within 7 days of harvesting the cells using the RiboPure RNA Purification Bacteria Kit (Ambion, Life Technologies Ltd, UK) according to manufacturers’ guidelines. Messenger RNA (mRNA) was enriched from the total RNA using the MICROBExpress bacterial mRNA enrichment kit (Ambion). Complimentary DNA (cDNA) libraries were prepared according to the manufacturer’s instructions, using the Illumina TruSeq Stranded mRNA kit and low sample protocol. The enriched mRNA samples were normalised to 100 ng and 5µl was added to 13 µl of the fragment prime finish mix to prepare sequencing libraries for each sample.