Pool RNA was isolated from four independently grown cultures of E. coli K-12 MG1655. The pool of control RNA was isolated from independent cultures of bacteria grown aerobically in the absence of nitrate or nitrite, but in the presence of TMAO. Test RNA from E.coli K-12 MG1655 fnr- was isolated from three independently anaerobically grown cultures plus nitrate. Samples were mixed with 2 volumes of RNA Protect (Qiagen Ltd) to stabilise the total RNA. An RNeasy kit (Qiagen Ltd) was used to extract the total RNA according to manufacturers’ instructions. Contaminating DNA was removed by using on-column DNase I digestion (Qiagen Ltd). The quality and quantity of the RNA preparations were determined with an Agilent 2100 Bioanalyzer by using the RNA 6000 nano assay Labchip (Agilent, Stockport, United Kingdom).
