title: GSM6568498: F2; Clostridioides difficile; OTHER
library_name: GSM6568498
library_construction_protocol: RIL-seq was performed, following the original protocol published by Melamed and collogues with minor alterations. Briefly, C. difficile 630 WT and hfq::3XFLAG were grown in sterile filtered TY in four biological replicates until OD600 of ∼1.2. Of each replicate, 100 ODs were harvested (4,500 x g, 15min at 4 °C), resuspended in 10 ml ice-cold 1xPBS and irradiated with UV light (254 nm, 80,000 mJ/cm2). Following centrifugation (4,500 x g, 15min at 4 °C), pellets were resuspended in 800 µl of ice-cold wash buffer (50 mM NaH2PO4, 300 mM NaCl, 0.05% Tween, 1:200 diluted protease inhibitor cocktail set III (Calbiochem)) supplemented with 0.1 U/μL of recombinant RNase inhibitor (wash buffer-RIn, Takara). Mechanical cell lysis was achieved by mixing each sample with 0.1 mm glass beads and grinding in a Retsch mixer mill at a frequency of 30/s for 5 min. The grinding was repeated four times, after each step the adaptors were place on ice for 2 min. The resulting supernatant was transferred to a new tube (17,000 x g, 2 min at 4 °C), 400 µl wash buffer-RIn was added to the glass beads and the grinding was repeated once more. For Hfq co-immunoprecipitations, the accumulated lysates were incubated with protein A/G magnetic beads (Thermo-Fisher) pre-coupled with anti-Flag antibody (M2 monoclonal antibody, Sigma-Aldrich) for 2 h at 4°C with rotation, followed by three washing steps with wash buffer-RIn. Next, samples were treated with 480 µl of wash buffer supplemented with with RNase A/T1 (1:520, Thermo Fisher Scientific) for 5 min at 22°C, trimming any exposed RNA ends. The process was stopped by washing each sample with 200 µl of wash buffer supplemented with SUPERase In RNase inhibitor (final concentration of 0.1 U/μL, Ambion) three times for 5 min at 4 °C. Following PNK treatment for 2 h at 22 °C (1x PNK buffer A, 1 mM ATP, 1 U/µl recombinant RNAse inhibitor, 0.5 U/µl T4 PNK (New England BioLabs)), samples were washed again three times with wash buffer-RIn. Hfq-bound RNAs were then proximity ligated with T4 RNA ligase I (1x T4 RNA ligase buffer, 9% DMSO, 1 mM ATP, 20% PEG 8000, 0.6 U/µl recombinant RNase inhibitor, 2.7 U/µl T4 RNA ligase I (New England BioLabs)) overnight at 22°C with agitation, followed by 3 washing steps with wash buffer-RIn at 4 °C. Finally, RNA was eluted by incubating the beads with Proteinase K (50 mM Tris-HCl pH 7.8, 50 mM NaCl, 1% SDS, 5 mM EDTA pH 8.0, 5 mM β-mercaptoethanol, 0.1 U/µl recombinant RNase inhibitor, 0.33 mg/ml Proteinase K (Thermo Fisher Scientific)) for 2 h at 55 °C. RNA purification was achieved using TRIzol LS according to the manufacturer's instructions.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. cDNA library preparation and sequencing were performed by Vertis Biotechnologie AG. First, oligonucleotide adapters were ligated to the RNA 5' and 3' ends followed by first-strand cDNA synthesis using M-MLV reverse transcriptase and the 3' adapters as primers. The resulting cDNAs were PCR-amplified using a high fidelity DNA polymerase for 14-16 cycles. Next, cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and quality controlled by capillary electrophoresis on a Shimadzu MultiNA microchip electrophoresis system. For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The resulting cDNA pool was then size fractionated in the size range of 170 – 400 bp by polyacrylamide gel electrophoresis and paired-end sequenced on an Illumina HighSeq system using 2 x 150 bp read lengths. RNA-seq (size fractionation)