title: GSM7868275: Growth_dkhpA_M_2; 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 20min, 4500rpm at 4°C and the supernatant was completely discarded. Cells were suspended in 600μl of 10mg/ml lysozyme in TE buffer (pH8.0) and incubated at 37°C for 10min. Next, 60μl of 10% w/v SDS was added and everything mixed by inversion. Samples were incubated in a water bath at 64°C, 1-2min before adding 66μl 3MNaOAc, pH5.2. Next, 750μl acid phenol (Roti-Aqua phenol) was added, followed by incubation for 5min. at 64°C. Samples were briefly placed on ice to cool before centrifugation for 15min, 13,000rpm at 4°C. The aqueous layer was transferred into a 2ml phase lock gel tube (Eppendorf), 750μl chloroform (Roth, #Y015.2) was added, and everything centrifuged for 12min, 13,000rpm at room temperature. For ethanol precipitation, the aqueous layer was transferred to a fresh tube, 2 volumes of 30:1 mix (EtOH:3MNaOAc, pH6.5) 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 50µl RNase-free water and stored at -80°C. For preparation cDNA library of the immunoprecipitated RNA samples, NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) was used. Briefly, 3.5 μl of RNA was mixed with 1 μl of 3' SR Adaptor (diluted 1:10 in nuclease-free water) and incubated in a preheated thermal cycler for 2 min at 70 °C.  The samples were transferred to ice and added with the premixed 3' ligation mix (5 μl 3' Ligation Reaction Buffer, 1.5 μl 3' Ligation Enzyme Mix). The ligation mixture was then incubated for 1 h at 25 °C. 0.25 μl of SR RT Primer and 2.5 μl nuclease-free water were added to the mixture and incubated at 75 °C for 5 min, 37 °C for 15 min and followed by 25 °C for 15 min. To ligate the 5' SR adaptor, the 5' ligation mixture (0.5 μl 1:10 diluted 5' SR Adaptor, 0.5 μl 10x 5' ligation reaction buffer, 1.25 μl 5' ligation enzyme mix) was added to each sample and incubated for 1 h at 25 °C. For synthesis first strand of cDNA, reverse transcription mix (4 μl first strand synthesis reaction buffer, 0.5 μl murine RNase inhibitor, 0.5 μl M-MLV reverse transcriptase) was added to each reaction and incubated for 1 h at 50 °C followed by 15 min at 70 °C to inactivate the reverse transcription reaction. 5 μl of cDNA was then proceeded to PCR amplification with the PCR mix (12.5 μl LongAmp Taq 2x master mix, 0.625 μl SR primer, 6.25 μl nuclease-free water) and 0.625 μl index (X) primer. PCR program (30s at 94°C, 15 s at 94 °C, 30 s at 62 °C and 15 s at 70 °C for 19 cycles, 5 s at 70 °C) was carried out. PCR products were mixed with 25 μl of GLII buffer and loaded on 6 % polyAcrylamide gel containing 8 M urea. After staining the gel for 10 min in 1x TBE buffer with a 1:10,000 dilution of SYBR Gold Nucleic Acid Gel Stain (Invitrogen), the bands in the range of 130- 1000 bp were cut into small pieces and transfer to a 2 ml LoBind tube. 500 μl of DNA elution buffer was added and incubated at the room temperature for 2 h. After transferring the elute and the gel debris to centrifuge tube filters (Sigma) and centrifugation (5000 rpm, 1 min), the elute was recovered by adding 1 μl liner acrylamide (NEB) and precipitating in ethanol at -80°C for 1 h. After precipitation, the pellet was washed with 80% ethanol and resuspended in 12 μl nuclease free water (NEB). 1 μl of the prepared cDNA library was subjected to Bioanalyzer analysis.   Amplified cDNAs were pooled and sequenced on an illumine NextSeq 2000 platform by the CoreUnit Sysmed from the University of Wuerzburg.