Analysis of gene expression in Porphyromonas gingivalis hemH and ihtB deletion mutant strains grown under heme- and iron-replete conditions (RNA-seq analysis)
Analysis of gene expression in Porphyromonas gingivalis hemH and ihtB deletion mutant strains grown under heme- and iron-replete conditions (RNA-seq analysis)
After cultivation, cells were harvested from 2 mL cultures by centrifugation (8,000×g, 10 min, 4°C), and the bacterial pellet was resuspended in 20 µL of 75% ethanol, immediately frozen in liquid nitrogen, and stored at -80℃.  P. gingivalis strains were cultured at 37℃ in anaerobic conditions (80% N2, 10% CO2, 10% H2) on Schaedler anaerobe blood agar (ABA) plates supplemented with 5% sheep blood and vitamin K3 (Argenta, Poznań, Poland) for 3-5 days, and then in a liquid basal medium (BM) composed of 3% trypticase soy broth (Becton Dickinson, Sparks, MD, USA), 0.5% yeast extract (Biomaxima), 0.05 mg/L vitamin K3 (Fluka, Munich, Germany), and 0.05% L-cysteine (Sigma-Aldrich, St. Louis, MO, USA). To grow bacteria in iron and heme-rich conditions, BM was supplemented with 7.7 μM hemin chloride (Pol-Aura, Morąg, Poland) (BM+Hm). Bacteria were grown for 3 passages in Bm+Hm medium. In the final passage, a culture was established in BM+Hm at OD₆₀₀ 0.2 and incubated for ~6 h, reaching an OD₆₀₀ of approximately 0.6-0.7. Total RNA isolation was performed by Novogene (Cambridge, United Kingdom) according to the company standard protocols. Firstly, ribosomal RNA was removed from total RNA, followed by ethanol precipitation. After fragmentation, the first strand cDNA was synthesized using random hexamer primers. During the second strand cDNA synthesis, dUTPs were replaced with dTTPs in the reaction buffer. The directional library was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. 