title: GSM5745055: ID_006657_FoxI_ctrl_B; Fusobacterium nucleatum subsp. nucleatum ATCC 23726; RNA-Seq
library_construction_protocol: Total RNA was extracted using the hot phenol protocol. Bacterial cultures were grown to the desired OD600, mixed with 0.2 volumes of STOP solution (95 % ethanol, 5 % phenol) and snap-frozen at – 80 °C if not directly processed. The bacterial solution was centrifuged for 20 min, 4500 rpm at 4 °C and the supernatant was completely discarded. Cells were suspended in 600 μl of 10 mg/ml lysozyme in TE buffer (pH 8.0) and incubated at 37 °C for 10 min. Next, 60 μl of 10 % w/v SDS was added and everything mixed by inversion. Samples were incubated in a water bath at 64 °C, 1-2 min before adding 66 μl 3 M NaOAc, pH 5.2. Next, 750 μl acid phenol (Roti-Aqua phenol) was added, followed by incubation for 5 min. at 64 °C. Samples were briefly placed on ice to cool before centrifugation for 15 min, 13,000 rpm at 4 °C. The aqueous layer was transferred into a 2 ml phase lock gel tube (Eppendorf), 750 μl chloroform (Roth, #Y015.2) was added, and everything centrifuged for 12 min, 13,000 rpm at room temperature. For ethanol precipitation, the aqueous layer was transferred to a fresh tube, 2 volumes of 30:1 mix (EtOH:3 M NaOAc, pH 6.5) was added and incubated overnight at -20 °C. Precipitated RNA was harvested by centrifugation, washed with cold 75 % v/v ethanol and air-dried. Purified RNA was resuspended in 50 µl RNase-free water and stored at -80 °C. The cDNA library preparation was carried by Vertis (Munich, Germany). The first step consisted out of rRNA removal. The rRNA depleted RNA was then fragmented via ultrasound (1 pulse of 30 seconds; 4 °C). The fragmented RNA was then used to ligate an adapter to the 3' end of the molecules. For first-strand cDNA synthesis, the M-MLV reverse transcriptase was used in conjunction with the introduced 3'-adapter serving as a primer. After purification, 5' Illumina TruSeq sequencing adapters were ligated to the cDNA. The resulting cDNA was used as input for during PCR amplification (10 – 20 ng µl-1). The amplified cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and further evaluated via capillary electrophoresis. The cDNA was pooled further purified to include only cDNA from 200 – 600 bp using a preparative agarose gel. The finished pooled libraries were sequenced by the Core unit SysMed (University of Würzburg) using an Illumina NextSeq 500 system and 75 bp read length.