title: GSM5048036: FLAG-3_S44_R1_001; Clostridioides difficile; RIP-Seq
library_construction_protocol: Pellets were resuspended in 800 µl lysis buffer (20 mM Tris-HCl (pH 8.0), 1 mM MgCl2, 150 mM KCl, 1 mM DTT) and 1 µl DNase I (Fermentas, 1 U/µl) was added to each sample. Cells were mixed with 800 µl of 0.1 mm glass beads and lysed in a RETSCH's Mixer Mill (30 Hz, 10 min, 4°C). Lysates were cleared by centrifugation for 10 min at 14,000 g and 4 °C. The supernatant was transferred to a new tube and the volume was adjusted to 900 µl with lysis buffer.25 µl of anti-FLAG antibody (clone M2, Merck/Sigma-Aldrich #F1804) was added to each sample before rocking at 4 °C for 1 hour. In the meantime, 75 µl Protein A Sepharose beads (Merck/Sigma-Aldrich #P6649) per sample were washed 3x with 1 ml lysis buffer. The lysates were added to the beads and rocked for another hour. Beads were washed 5x with 500 µl lysis buffer and finally resuspended in 550 µl lysis buffer. 50 µl of beads were transferred to a fresh tube and mixed with 1x protein loading buffer for Western blot analysis. 500 µl Phenol:Chloroform:Isoamylalcohol (25:24:1, pH 4.5, Roth) was added to the beads. The solution was mixed for 20 s, transferred to phase lock tubes (Eppendorf) and incubated at room temperature for 3 min. Tubes were spun for 30 min at 15,000 g and 15 °C. The aqueous phase was transferred to a new tube, 3 µl Glycoblue (Thermo Fisher Scientific) and 800 µl isopropanol was added. RNA precipitation was done overnight at -20 °C. Samples were centrifuged for 45 min at 15,200 rpm and 4 °C. Supernatant was discarded. RNA was washed 1x with 80% Ethanol and 1x with 100% Ethanol (centrifugation for 10 min, 15,200 rpm, 4 °C). The pellets were air-dried at room temperature and resuspended in 15.5 µl nuclease free water (65 °C, 5 min, 600 rpm). For DNAse I treatment, 2 µl DNase I (Thermo Fisher Scientific), 0.5 µl RNase inhibitor and 2 µl DNase buffer (Thermo Fisher Scientific ) was added to each sample. Incubation was done for 30 min at 37 °C. To isolate the RNA, 100 µl nuclease free water and 120 µl Phenol:Chloroform:Isoamylalcohol (25:24:1, pH 4.5, Roth) was added to the samples. RNA isolation and precipitation was done as described before. RNA was resuspended in 20 µl nuclease free water. RNA-sequencing was conducted by Vertis Biotechnology AG. In brief, quality of RNA samples was checked on a Shimadzu MultiNA microchip electrophoresis system. Fragmentation was done using ultrasound (4 pulses of 30 sec each at 4 °C). Adapter was ligated to the 3'end. First-strand cDNA synthesis was performed using M-MLV transcriptase and the 3' adapter as primer. cDNA was purified and 5' Illumina TruSeq adapter was ligated to the 3' end of the antisense strand. Amplification was done via PCR to 10-20 ng/µl using high fidelity protocol and 11 PCR cycles. The cDNA was then purified with Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis as stated above. For sequencing, the samples were pooled in equimolar amounts and sequenced on a Illumina NextSeq 500 system using 75 bp read length.