title: GSM7868295: RIP_C_BF_2_R1; Fusobacterium nucleatum subsp. nucleatum ATCC 23726; RIP-Seq
library_construction_protocol: RNA coimmunoprecipitation was performed as previously carried out for Hfq or ProQ (Chao et al., 2012; Smirnov et al., 2016). Briefly, bacterial cultures (4 replicates) were grown to mid-exponential phase (OD=0.5) and a volume of cells equal to 100 ODs was collected. Cells were suspended in 800 μl lysis buffer (20 mM Tris pH 8.0, 150 mM KCl, 1 mM MgCl2,1mM DTT) containing 8 μl of DNase I. The resuspended cells were added to a fresh 2ml tube with 800 μl of 0.1 mm glass beads and lysed at 30 Hz for 10 min using Retsch MM200. A volume of supernatant equivalent to 0.5 OD600 cells was added to 90 μl 1x protein loading dye and used for input control. 25 μl of Flag antibody (Monoclonal ANTI-FLAG M2, Sigma, #F 1804) was added to the supernatant and rotated for 30 min at 4 °C. The sample was then added with 75 μl pre-washed Protein A Sepharose (#P6649, Sigma) and incubated at 4 °C for 30 min with rotation. Afterwards, the beads were spun down and a volume equivalent to 0.5 OD600 cells were added to 90 μl 1x protein loading dye and used for flowthrough control. The beads were then washed 5 times with the lysis buffer by inverting the tubes and centrifuged roughly. The last washing was collected in 1x protein loading dye. After washing, resuspended the beads in 532 μl lysis buffer and collected 32 μl to 8 μl 5x protein loading dye for later western blot analysis. The remaining beads were added 500 μl P: C: I for RNA precipitation. After precipitation, the RNA pellets were resuspended in H2O and further subjected to DNase I digestion and purified by P: C: I. At the end, the RNA pellets were resuspended in 15 μl H2O and used 15 μl from one replicate for northern blot verification and 3.5 μl of other three replicates for the cDNA library preparation. 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.