title: GSM5048049: S4-KO_S10_R1_001; Clostridioides difficile; RNA-Seq
library_construction_protocol: 5 OD per culture were harvested by subsequent addition of 1/5 volume ice-cold stop mix (95% EtOH, 5% phenol). Tubes were immediately frozen with liquid nitrogen and stored at -80 °C. RNA was extracted via hot phenol extraction. The mixture was slowly thawed on ice and centrifuged for 20 min at 4500 g and 4 °C. The supernatant was completely discarded. Cells were resuspended in 600 µl lysozyme (10 mg/ml) in TE buffer (pH 8.0) and incubated for 10 min at 37 °C. 60 µl w/v SDS was added and mixed by inversion. The samples were heated to 64 °C in a water bath for 1-2 minutes. Subsequently, 66 µl 3 M sodium acetate (pH 5.2) and 750 µl acid phenol (Roti Aqua Phenol, Roth, #A980.1) was added. The mixture was incubated for another 5 min at 64 °C. Samples were cooled down on ice before centrifugation for 15 min at 13,000 rpm and 4 °C. The aqueous layer was transferred to a phase lock gel tube (Eppendorf). 750 µl chloroform (Roth, #Y015.2) was added before centrifugation for 12 min at 13,000 rpm at room temperature. The aqueous layer was transferred to a new tube and 2 volumes of EtOH:3 M NaOAc (30:1, pH 5.2) was added. RNA precipitation was done overnight at -20 °C. RNA was pelleted by centrifugation for 45 mins, 16,000 rpm at 4°C. Pellet was washed twice with EtOH (80% EtOH, 100% EtOH) and air-dried. RNA was resuspended in 50 µl nuclease free water (65 °C, 1000 rpm, 5 min). DNA contaminations were removed via DNase I digestion as described previously above. 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 (1 pulse 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 14 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.