title: GSM4905303: ID-005362_FNP_E_untreated_repA; Fusobacterium nucleatum subsp. polymorphum ATCC 10953; 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. Library preparation for dRNA-seq was accomplished by Vertis Biotechnology AG. In brief, total RNA was analyzed on a Shimadzu MultiNA microchip electrophoresis system. 23S/16S ratio for all samples was 1.3. RNA was fragmented via ultrasound (4 pulses a 30 sec, 4 °C) and subsequently treated with T4 Polynucleotide Kinase (NEB). Half of the samples were then treated with terminator exonuclease (TEX) for dRNA-seq. For the cDNA synthesis, the RNA fragments were poly(A)-tailed and 5'PPP structures were removed with RNA 5' Polyphosphatase (Epicentre). The RNA sequencing adapter with the barcodes were ligated to the 5'-monophosphate of the fragments. First strand cDNA was synthesized with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. Amplification of cDNA was done via PCR to an approximate amount of 10 20 ng/µl. cDNA was purified with the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed with Shimadzu MultiNA microchip electrophoresis. Equimolar amounts of the samples were pooled for sequencing. cDNAs had a size between 200 and 550 bp. The library pool was fractionated via differential clean-up with the Agencourt AMPure kit. The cDNA pool was checked with capillary electrophoresis as stated above. Libraries were sequenced on an Illumina NextSeq 500 system using 75 bp read length.