title: Analysis of gene expression in Porphyromonas gingivalis hmuS deletion mutant strain (RNA-seq analysis)
design: Analysis of gene expression in Porphyromonas gingivalis hmuS deletion mutant strain (RNA-seq analysis)
library_construction_protocol: 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 W83 wild-type (WT), and hmuS deltetion mutant (ΔhmuS) strain constructed in W83 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 ~20 h. 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. 