Bacterial RNA Extraction Protocol

After harvesting the bacteria by centrifuging the cultures at 4800× g for 10 min, the bacterial pellet was suspended in 500 µL of ice-cold phosphate buffer solution (PBS). The suspension was then centrifuged at 500× g for 10 min at 4 °C. This washing step was repeated twice. The RNA was extracted by a combination of the CTAB and QIAGEN RNeasy Mini Kit methods. In short, CTAB/PVP/BME solution was added to the bacteria pellet and, after resuspension, subjected to three cycles of deep freezing at −80 °C, followed by thawing at room temperature to allow the lysing of the cells, and was subsequently processed as described by Jordon-Thaden et al. [27 (link)], albeit after DNAse treatment with a TURBO DNA-free kit, a phenol/chloroform/IAA step was included, followed by two cycles of chloroform/IAA extractions. For precipitation, a 1/20th volume of 5 M NaCl and two volumes of ethanol were added, and then the solution was incubated for 20 min at −20 °C. RNA purification was carried out with Qiagen spin columns, washing with Qiagen washing solutions, and finally elution with 50 microliters of RNase-free water. The quantity and purity of the total RNA samples were assessed by ultraviolet spectroscopy using a DS-11 spectrophotometer (DeNovix, Inc., Wilmington, North Carolina, USA).