Transcriptome Analysis of Neisseria gonorrhoeae

Total RNA was isolated from urethral swab and cervicovaginal lavage specimens using TRIzol (Invitrogen) as described previously (38 (link)). Briefly, specimens were washed twice with 70% ethanol and treated with Turbo DNase (Ambion), the RiboMinus kit (Invitrogen) (to deplete eukaryotic rRNA), and the Microbe Express kit (Ambion) (to deplete bacterial rRNA) using diethylpyrocarbonate (DEPC)-treated EDTA-free water. Gonococcal isolates that corresponded to each specimen were grown overnight on chocolate agar plates at 37°C in 5% CO₂ prior to inoculation in chemically defined medium (CDM) containing ferric nitrate (100 µM). Liquid cultures were inoculated at an optical density at 600 nm (OD₆₀₀) of 0.1, harvested after 3 h, and pelleted, and RNA was extracted as described above.