GSM4696500: RNAtag-seq_LE-TY_3; Clostridioides difficile 630; RNA-Seq
dRNA-seq and RNAtag-seq: Total RNA was extracted using the hot phenol protocol. Bacterial cultures were grown to the desired OD600, mixed with 0.2 volumes of STOP solution (95 % ethanol, 5 % phenol) and snap-frozen at – 80 °C if not directly processed. The bacterial solution was centrifuged for 20 min, 4500 rpm at 4 °C and the supernatant was completely discarded. Cells were suspended in 600 μl of 10 mg/ml lysozyme in TE buffer (pH 8.0) and incubated at 37 °C for 10 min. Next, 60 μl of 10 % w/v SDS was added and everything mixed by inversion. Samples were incubated in a water bath at 64 °C, 1-2 min before adding 66 μl 3 M NaOAc, pH 5.2. Next, 750 μl acid phenol (Roti-Aqua phenol) was added, followed by incubation for 5 min. at 64 °C. Samples were briefly placed on ice to cool before centrifugation for 15 min, 13,000 rpm at 4 °C. The aqueous layer was transferred into a 2 ml phase lock gel tube (Eppendorf), 750 μl chloroform (Roth, #Y015.2) was added, and everything centrifuged for 12 min, 13,000 rpm at room temperature. For ethanol precipitation, the aqueous layer was transferred to a fresh tube, 2 volumes of 30:1 mix (EtOH:3 M NaOAc, pH 6.5) was added and incubated overnight at -20 °C. Precipitated RNA was harvested by centrifugation, washed with cold 75 % v/v ethanol and air-dried. Purified RNA was resuspended in 50 µl RNase-free water and stored at -80 °C. RIP-seq: For each condition, 50 OD were harvested by centrifugation for 20 min at 4000 rpm at 4 °C, snap frozen and resuspended in 800 µl lysis buffer (20 mM TRIS pH 8.0, 1 mM MgCl2, 150 mM KCl, 1 mM DTT). Subsequently, each sample was mixed with 1 µl DNase I (Fermentas, 1U/µl) and 800 µl of 0.1 mm glass beads and lysed in a RETSCH's Mixer Mill (30 Hz, 10 min, 4°C). Bacterial lysates were cleared by centrifugation for 10 min at 14,000 x g and 4 °C. Approximately 900 µl supernatant was transferred to a new tube and incubated with 25 µl (1/2 * OD in µl) of mouse-anti-FLAG antibody (clone M2, Merck/Sigma-Aldrich #F1804) at 4 °C for 45 min (rocking). Immunoprecipitation of FLAG-tagged Hfq was performed by incubating each sample with 75 µl pre-washed (3 x resuspended in 1 ml lysis buffer and centrifuged at 10,000 rpm for 1 min) protein A sepharose beads (Merck/Sigma-Aldrich #P6649) for 45 min at 4 °C (rocking). Beads and captured proteins were washed 5 x with 500 µl lysis buffer (mixed by inversion and centrifuged at 10,000 rpm for 1 min) and resuspended in 500 µl lysis buffer. Finally, RNA, co-immunoprecipitated with antibodies and protein A sepharose beads, was eluted by adding the same volume of Phenol:Chloroform:Isoamylalcohol (25:24:1, pH 4.5, Roth). The solution was mixed for 20 s, transferred to PLG tubes (Eppendorf) and incubated at room temperature for 3 min. Following centrifugation (30 min, 15,200 x g, 15 °C) the aqueous phase was transferred to new tubes and RNA was precipitated over night at -°20 °C by adding 30 µg Glycoblue and 800 µl isopropanol. Precipitated RNA was pelleted (centrifugation for 45 min, 15,200 rpm, 4 °C), washed with 80 % and 100 % ethanol (centrifugation for 10 min, 15,200 rpm, 4 °C) and resuspended in 15,5 µl nuclease-free water (65 °C, 1 min, 600 rpm). For DNase treatment, purified RNA was incubated with 2 µl DNase I, 0.5 µl RNase inhibitor and 2 µl 10 x DNase buffer for 30 min at 37 C. To each sample, 100 µl nuclease free water was added and DNase treated RNA was purified by Phenol:Chloroform:Isoamylalcohol treatment, as described before. Precipitation was performed overnight at – 20 °C by mixing the samples with 3 x the volume of sodium-acetate/ethanol (1:30), followed by centrifugation and washing with 80 % and 100 % ethanol. Finally, air-dried pellets were resuspended in 20 µl nuclease-free water (65 °C, 1 min, 600 rpm). dRNA-seq: Library preparation for dRNA-seq was accomplished by Vertis Biotechnology AG. In brief, total RNA was analyzed on a Shimadzu MultiNA microchip electrophoresis system. 23S/16S ratio for all samples was 1.3. RNA was fragmented via ultrasound (4 pulses a 30 sec, 4 °C) and subsequently treated with T4 Polynucleotide Kinase (NEB). Half of the samples were then treated with terminator exonuclease (TEX) for dRNA-seq. For the cDNA synthesis, the RNA fragments were poly(A)-tailed and 5'PPP structures were removed with RNA 5' Polyphosphatase (Epicentre). The RNA sequencing adapter with the barcodes were ligated to the 5'-monophosphate of the fragments. First strand cDNA was synthesized with an oligo(dT)-adapter primer and M-MLV reverse transcriptase. Amplification of cDNA was done via PCR to an approximate amount of 10 20 ng/µl. cDNA was purified with the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed with Shimadzu MultiNA microchip electrophoresis. Equimolar amounts of the samples were pooled for sequencing. cDNAs had a size between 200 and 550 bp. The library pool was fractionated via differential clean-up with the Agencourt AMPure kit. The cDNA pool was checked with capillary electrophoresis as stated above. Libraries were sequenced on an Illumina NextSeq 500 system using 75 bp read length. RNAtag-seq: Total RNA quality was checked using a 2100 Bioanalyzer with the RNA 6000 Nano kit (Agilent Technologies) and rRNA was detected. Equal amounts of samples (~ 500 ng) were used for the preparation of cDNA libraries with the RNA-tag-Seq protocol as previously published by Shishkin et al. (Shishkin et al., 2015) with minor modifications. Briefly, the RNA samples were fragmented with FastAP buffer at 94˚C for 3 min and were dephoshorylated using the FastAP enzyme (Thermo Scientific) at 37 ˚C for 30 min followed by bead purification with 2 volumes of Agencourt RNAClean XP beads. Fragmentation profiles were checked using RNA 6000 pico kit (Agilent) with a 2100 Bioanalyzer. The RNA fragments were ligated with 3´- barcoded adaptors at 22˚C for 1 hour and 30 min using T4 RNA Ligase (NEB). Barcoded RNA samples were pooled together, the ligase was inhibited by RTL buffer (Qiagen RNeasy Min Elute Cleanup Kit) and purified with RNA Clean & Concentrator-5 column (Zymo). rRNA were depleted from pools using Ribo-Zero (Bacteria) Kit (Illumina) and purified with RNA Clean & Concentrator-5 column (Zymo). The profiles before and after rRNA depletion were analysed with RNA 6000 pico kit (Agilent) with a 2100 Bioanalyzer. The rRNA depleted RNA were reverse transcripted and the first strand of the cDNA were synthsized using a custom AR2 oligo (Sigma-Aldrich) and the AffinityScript multiple temperature cDNA synthesis kit (Agilent) at 55°C for 55 minutes. The RNA was degraded with 1M NaOH at 70 °C for 12 minutes, the reactions were neutralized with Acetic acid and were purified with 2 volumes of MagSi-NGSPREP Plus beads (AMSBIO). A second 3Tr3 adaptor were ligated to the cDNA using T4 RNA Ligase (NEB) overnight at 22°C followed with two bead purification steps with 2 volumes of MagSi-NGSPREP Plus beads (AMSBIO). A PCR enrichment test was performed in order to determine the number of PCR cycles that are nesessery for each pool followed by a bead purification step and a QC with DNA HS kit (Agilent). Then, the final PCR was performed with the number of cycles determined from the previous step using P5 and P7 primers. Two sequential bead purification steps were performed with 1.5 and 0.7 respectively. Libraries were quantified with the Qubit 3.0 Fluometer (ThermoFisher) and the library quality and size distribution (~480 bp peak size) was checked using a 2100 Bioanalyzer with the High Sensitivity DNA kit (Agilent). Sequencing of pooled libraries, spiked with 5% PhiX control library, was performed with ~8 million reads / sample in single-end mode on the NextSeq 500 platform (Illumina) with the High Output Kit v2.5 (75 Cycles). RIP-seq: RNA quality was controlled using a 2100 Bioanalyze