Transcriptomic Analysis of E. coli MG1655

E. coli MG1655 was obtained from the ATCC. MG1655 ΔnudC:Kan was previously reported (Bird et al. 2016 (link)). E. coli MG1655 or MG1655 ΔnudC:Kan cells were grown overnight and then back diluted 1:100 in 25 mL LB (10 g Bacto-tryptone, 5 g Bacto-yeast extract and 10 g NaCl per liter) containing 25 µg mL^–1 kanamycin (only for the ΔnudC:Kan strain) in 125 mL flasks (Bellco). Cultures were shaken at 220 rpm at 37°C. When cell density reached an OD₆₀₀ ∼ 3.4 (∼8 h), 5 mL of the cell suspension was centrifuged (1 min, 10,000g at room temperature) to collect cells, supernatants were removed, and cell pellets were rapidly frozen on dry ice. To extract total RNA, frozen pellets were resuspended in 1 ml of TRI Reagent solution (Molecular Research Center). Samples were incubated at 70°C for 10 min and then centrifuged (10 min, 21,000g, 4°C) to remove insoluble material. The supernatant was transferred to a fresh tube and 200 µL of chloroform was added, samples were mixed by vortexing, and then centrifuged (10 min, 21,000g, 4°C). The aqueous phase was transferred to a fresh tube, extracted with acid phenol:chloroform twice (Ambion), and RNA transcripts were recovered by ethanol precipitation, washed with 75% ethanol at 4°C and resuspended in RNase free water (Ambion).