title: GSM5048016: Grad_01_Fraction_04_Rep_1; Clostridioides difficile; RNA-Seq
library_construction_protocol: Glycerol gradient fractionation was performed as previously described (Hor et al., 2020). C. difficile was grown in 2x100 ml BHI to late-exponential phase (OD 1.6). The cultures were cooled down for 15 minutes on ice-water slurry before harvesting by centrifugation for 20 min at 4 °C and 4,000 g. Pellets were washed three times with ice-cold 1x TBS before resuspension in 500 µl ice-cold 1x lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM, KCl, 1 mM DTT, 1 mM PMSF, 0.2% Triton X 100, 20 U/ml DNase I (Thermo Fisher Scientific), 200 U/ml RNase inhibitor. Cell lysis was performed in 2 ml tubes containing Lysing matrix E (MP Biomedicals, 1.4 mm ceramic spheres, 0.1 mm silica spheres and one 4 mm glass bead) by using a FastPrep-24 (MP Biomedicals) machine at 6 m/s for 30 seconds. The lysate was centrifuged for 30 minutes at 4 °C and 4,000 g to remove beads and debris. For input controls 10 µl lysate was mixed with 1 ml TRIzol and 20 µl was mixed with 5x protein loading buffer. The 10 – 40% (w/v) glycerol gradient (in lysis buffer without DNase I or RNase inhibitor) was formed in an open-top polyallomer ultracentrifugation tube (Seton Scientific) by using a Gradient Station model 153 (Biocomp).  200 µl of the cleared cell lysate was layered on top of the gradient. The gradient was centrifuged for 17 h at 4 °C and 100,000 rcf (23,700 rpm) with an SW 40 Ti rotor (Beckman Coulter). Fractionation was done manually into 20 fractions with a volume of 590 µl.. For quality control, all fractions were analyzed with a NanoDrop (Thermo Fisher Scientific) Spectrophotometer at 260 nm. 90 µl of each fraction was mixed with 5x protein loading buffer to perform LC-MS/MS.500 µl of the fractions was used for total RNA isolation. Therefore, 50 µl 10% SDS (25 µl for the pellet) and 600 µl of acidic phenol/chloroform/isoamyl alcohol (P/C/I, Roth, 300 µl for the pellet) was added to the fractions. After mixing for 30 seconds the mixture was incubated for 5 minutes at room temperature. Additionally, 400 µl chloroform was added to the Trizol-dissolved lysate and mixed. The lysate was also incubated for 5 minutes at room temperature. Samples were centrifuged for 15 minutes at 4 °C and 13,000 rpm. The aqueous phase of all samples was collected. For RNA precipitation 1.4 ml ice-cold 30:1 Ethanol:3 M NaOAc (pH 6.5) and 1 µl GlycoBlue (Thermo Fisher Scientific) co-precipitant was added. RNA was precipitated over night at -20 °C. RNA was pelleted at 4 °C for 30 minutes and 13,000 rpm and washed with 70% ice-cold Ethanol, followed by centrifugation at 4 °C for 15 minutes and 13,000 rpm. The pellet was air-dryed and subsequently dissolved in 40 µl DEPC-H2O. To digest DNA 0.5 µl RNase inhibitor, 5 µl DNase I buffer with MgCl2 (Thermo Fisher Scientific), 4 µl DNase I (Thermo Fisher Scientific) and 0.5 µl DEPC-H2O was added to each RNA sample. Digestion was performed for 45 minutes at 37 °C. RNA was again purified as described above. This time 150 µl DEPC-H2O and 200 µl acidic P/C/I was added to the samples. RNA was finally dissolved in 35 µl DEPC-H2O and stored at -80 °C. 5 µl of the gradient samples were diluted in 45 µl DEPC-H2O. 10 µl of the diluted RNA was mixed with a 1:100 dilution of ERCC spike-in mix 1 (Thermo Fisher Scientific). Library preparation and sequencing was carried out by Vertis Biotechnology AG. In brief, the RNA was fragmented using ultrasound (4 pulses of 30 seconds each at 4 °C) followed by 3' adapter ligation. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3'-adapter as primer. After purification of the cDNA the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. Amplification of cDNA (10-20 ng/µl) was done via PCR using high fidelity RNA polymerase and 12 sequencing cycles. cDNA purification was performed with Agencourt AMPure XP kit (Beckman Coulter Genomics). The samples were pooled for sequencing. cDNA pool was eluted in the size range of 200 – 500 bp from a preparative agarose gel. Quality check of RNA was performed in between steps with capillary electrophoresis using a Shimadzu MultiNA microchip. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.