GSM5124411: F. nucleatum delta-carR Rep 1; Fusobacterium nucleatum subsp. nucleatum; RNA-Seq
F. nucleatum RNA was extracted using RNeasy Mini Kits (Qiagen) according to the manufacture's protocol. Fusobacterial cell pellets from 3 ml log-phase culture (OD600 ~ 0.6) were harvested and resuspended into 200 μl of chilled 10 mM RNA-free TE (10 mM Tris-HCl, pH 8; 1 mM EDTA). Each suspension was transferred to a fast-protein tube (Q BIOgene), which contained 700 μl RLT buffer (RNeasy Mini Kit, Qiagen) and 7 μL β-mercaptoethanol (BME) (ThermoFischer Scientific. Cells were lysed by using Ribolyser (Hybaid), and supernatants were obtained by centrifugation at 13000 x g for 5 min at 4°C. RNA was then purified from the supernatant accordingly, and purified RNA was treated with DNAse (Qiagen) and cleaned by using an RNeasy clean up kit (Qiagen). Quality of RNA samples was determined RNA Integrity Number (RIN) values greater than 8 using an Agilent 2100 Bioanalyzer (Agilent Technologies). 2 µg total RNA from F.nucleatum parent and mutant strains were used. Stable RNAs were depleted with the RiboZero Kit according to manufacturer's instructions (Epicentre). Afterwards the remaining mRNA was purified using RNA MinElute columns (Qiagen) and checked for quality with the Bioanalyzer (Agilent). Fragmentation of mRNA, reverse transcription to cDNA, adenylation of 3' ends, adapter ligation and PCR amplification were performed according to TrueSeq Stranded mRNA library instructions (Illumina). Prior to paired-end sequencing of the whole transcriptome cDNA libraries on an Illumina MiSeq, the quality and concentration of the libraries were checked using the Bioanalyzer (Agilent).