Cell culture and total RNA Isolation for 16S rRNA sequencing

E. coli strains BW25113 JW3718Δ and BW25113 JW2171Δ (strains hereafter referred to by gene deletion names RsmGΔ and RsuAΔ, respectively), deficient for 16S rRNA modifying enzymes RsmG and RsuA respectively, were purchased from the Keio Knockout collection [37] (GE Dharmacon). E. coli strains K12 MG1655, RsmGΔ, RsuAΔ and S. enterica strain LT2 were grown in LB media (supplemented with 50 μg/ml kanamycin for RsmGΔ and RsuAΔ) at 37°C to an A₆₀₀ = 0.8–1.0. Cells were harvested by centrifugation and total RNA was extracted with Trizol (Thermo Fisher) following the manufacturer’s recommended protocol. All total RNA samples were treated with DNase I (NEB) (2U/10 ug RNA) in the manufacturer's recommended buffer at 37°C for 15 minutes. Following the DNase I reaction, RNA was extracted by acid phenol/chloroform extraction (pH 4.4, Fisher Scientific) and two rounds of chloroform extraction. RNA was precipitated with sodium acetate (pH 5.2) and ethanol. RNA was resuspended in nuclease-free water and stored at -80°C. For experiments where human RNA was used as a background, total RNA was extracted from 10⁷ HEK 293T cells following the same steps.