Analysis of gene expression in Porphyromonas gingivalis hemH deletion mutant strain grown under heme-depleted conditions (RNA-seq analysis)
Analysis of gene expression in Porphyromonas gingivalis hemH deletion mutant strain grown under heme-depleted conditions (RNA-seq analysis)
1 ml of bacterial culture was centrifuged (8,000×g, 10 min, 4°C), and the bacterial pellet was resuspended in 800 μl of Fenozol (A&A Biotechnology, Gdańsk, Poland) and stored in -80°C P. gingivalis strains were cultured at 37℃ in anaerobic conditions (80% N2, 10% CO2, 10% H2) using in basal medium (BM) composed of 3% trypticase soy broth (Becton Dickinson), 0.5% yeast extract (Biomaxima) 0.05 mg/L vitamin K3 (Fluka), and 0.05% L-cysteine (Sigma-Aldrich). P. gingivalis was first cultured on BM agar plates supplemented with 7.7 µM heme for 4 days. Subsequently, the bacteria were transferred to liquid BM containing 2 µM heme, inoculated at an initial OD₆₀₀ of 0.2, and cultured for 24 h. Then, two 24-h passages in BM lacking heme, each initiated at an OD₆₀₀ equal to 0.2, were carried out. In the final step, a culture was established in heme-free BM at OD₆₀₀ 0.2 and incubated for 20 h, reaching an OD₆₀₀ of approximately 1–1.5. Total RNA was extracted from ~ 10^9 bacteria using the Total RNA Mini Kit (A&A Biotechnology, Gdańsk, Poland), followed by treatment with the Clean-up RNA Concentrator Kit (A&A Biotechnology) to eliminate residual genomic DNA contamination. RNA purity and integrity were evaluated using spectrophotometry and agarose gel electrophoresis, respectively. RNA samples were stored at –80 °C or in dry ice until further processing.  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. 