RNA-seq of E. coli treated
E. coli was cultured on LB agar media and incubated at 37 for 24-48 hours. Two to four pure colonies with similar morphologies for each strain were inoculated in 5 ml MRS and LB broth media, selective for each strain. The cultures were incubated for 16 hours in a shaking incubator till the mid-log phase (OD600 ~ 0.5-1) at 37 and 110 rpm speed for 24 hours. The cultures were then transferred at a 1:100 ratio into 5 mL of MRS and LB medium containing fluoride. Approximately 50 L of the homogenised bacterial suspension of both strains was inoculated into 4.95 mL of MRS medium and LB medium, as well as 4.95 mL of high- and low-fluoride media. The high (2500 mg/l NaF) and low fluoride (9 mg/l NaF) medium was prepared with MRS and LB media. The control culture with no fluoride and the low fluoride culture were incubated in a shaking incubator under similar conditions of temperature and speed mentioned above for 24 hours (until the culture reached an OD600 of approximately 1.0), whereas high fluoride cultures were incubated for only 6 hours to achieve optimum OD and get some bacterial pellet for RNA extraction. The optical density of the medium was determined using UVVis Spectrophotometer at a wavelength of 600 nm, following the methods of Li et al (2020) and Dutta et al (2020) (Li et al., 2020, Dutta et al., 2020). Cells in the suspension solution were collected by centrifugation for 10 minutes at 10,000 rpm at 4 , and RNA was extracted using Trizol reagent (Invitrogen, USA) according to the manufacturers instructions. RNA purity and concentration were measured using a Qubit, and the quality was checked by gel electrophoresis. The cDNA library was constructed with extracted high-quality mRNA (OD260/280 = 1.92.0, OD260/230  2.0) in accordance with the RNA sample prep kit (Illumina, USA).
SUB16183455
Ecoli_16_1