title: GSM8682291: FNN, H20, 16h, rep C; Fusobacterium nucleatum; RNA-Seq
library_construction_protocol: To isolate fusobacterial RNA, samples were incubated 1:1 (v:v) with RNAprotect Bacteria (Qiagen) for five minutes at RT, snap frozen in liquid nitrogen and stored at -80°C until further processing. To purify RNA the miRNeasy Micro kit (Qiagen) was used. Briefly, cell dilutions were thawed on ice, centrifuged at 4°C with 13,000 g for 20 minutes, the pellets resuspended in 100 μl 0.5 mg ml-1 lysozyme (Roth) in TE buffer pH 8.0 and incubated for two minutes at RT. The samples were then incubated with 700 μl Qiazol (Qiagen) for five minutes at RT and after addition of 160 μl chloroform shaken vigorously for 15 seconds to obtain phase separation. The upper aqueous phase was mixed 1:1.5 with 100% ethanol and loaded onto the Qiagen columns. Columns washes, DNase I digest and elution were performed according to the manual instructions. RNA concentrations were determined via NanoDrop measurements and samples were stored at -80°C until sequencing. RNA quality was investigated using the 2100 Bioanalyzer together with the RNA 6000 Pico kit (Agilent Technologies). Risbosomal RNA (rRNA) depletion was performing using the RiboCop META rRNA depletion kit (Lexogen) according to the manufacturer's instructions. Depleted RNA was further fragmented via ultrasound for 30 s at 4°C. After adapter ligation to the 3' end, the first-strand cDNA was synthetized using the M-MLV reverse transcriptase. Following purification, 5' Illumina TruSeq adapters were ligated to the cDNA, the cDNA amplified by PCR and the resulting product purified with the Agencourt AMPure XP kit (Beckman Coulter Genomics). After performing library quality control using the 2100 Bioanalyzer with the DNA High Sensitivity kit (Agilent Technologies), the cDNA was pooled, purified and sequenced using an Illlumina NextSeq 500 system with 10 million reads/sample in single end mode with 75 nt read length.