title: GSM2263200: ID-003338-01_Hfq_FLAG; Neisseria meningitidis 8013; RIP-Seq
library_construction_protocol: For dRNA-seq: N. meningitidis 8013 cells were grown in 50 ml tubes at 37 °C under vigorous shaking in GCBL++ medium and samples were collected by centrifugation at OD600 of 0.5 and 1.5, corresponding to the mid logarithmic and late logarithmic/early stationary growth phase, respectively. Transcription and translation were terminated by the addition of STOP Mix [95% (vol/vol) EtOH and 5% (vol/vol) phenol] and cells were frozen in liquid nitrogen. Samples were stored at −80 °C until RNA preparation. Frozen cell pellets were thawed on ice, and resuspended in a lysis solution consisting of 800 µl of 0.5 mg/ml lysozyme in TE buffer (pH 8.0) and 80 µl 10% SDS. Bacterial cells were lysed by placing the samples for 1–2 minutes at 65°C in a water bath. Total RNA was extracted from the lysates using the hot phenol method (Blomberg 1990). For RIP-seq:For each strain cells equivalent to an OD600 of 50 were collected in parallel and subjected to Hfq coIP and control coIP as described previously (Dugar et al. 2016). Cells were harvested by centrifugation at 6,000g for 20 min at 4 °C. Afterwards, cell pellets were resuspended in 1ml Lysis Buffer (20mM Tris-HCl, pH 8.0, 150mM KCl, 1mM MgCl2, 1mM dithiothreitol (DTT)) and subsequently centrifuged (5 min, 11,000g,4 °C). The pellets were shock-frozen in liquid nitrogen and stored at -80 °C. Frozen pellets were thawed on ice and resuspended in 0.8 ml Lysis Buffer. An equal volume of glass beads was then added to the cell suspension. Cells were then lysed using a Retsch MM40 ball mill (30 s-1, 10min) in pre-cooled blocks (4 °C) and centrifuged for 2min at 15,200g, 4 °C. The supernatant was transferred to a new tube, and an additional 0.4 ml of Lysis Buffer was added to the remaining un-lysed cells with beads. Lysis of the remaining cells was achieved by a second round of lysis at 30 s-1 for 5min. Centrifugation was repeated and this second supernatant was combined with the first one. The combined supernatant was centrifuged again for 30 min at 15,200g, 4 °C for clarification and the resulting supernatant (lysate fraction) was transferred to a new tube. The lysate was incubated with 35 ml anti-FLAG antibody (Monoclonal ANTI-FLAG M2, Sigma, #F1804) for 30 min at 4 °C on a rocker. Next, 75 ml of Protein A-Sepharose (Sigma, #P6649), prewashed with Buffer A, was added and the mixture was rocked for another 30 min at 4 °C. After centrifugation at 15,200g for 1min, the supernatant was removed. Pelleted beads were washed five times with 1 ml Lysis Buffer. Finally, 500 ml Lysis Buffer was added to the beads and RNA and proteins were separated by phenol-chloroform-isoamyl alcohol (ROTH 985.1) extraction and precipitated as described previously (Chao et al. 2012). For dRNA-seq: cDNA libraries of dRNAseq samples for were constructed by vertis Biotechnology AG, Germany (http://www.vertis-biotech.com/), as described previously for eukaryotic microRNA (Berezikov et al. 2006), but omitting the RNA size-fractionation step prior to cDNA synthesis. In brief, total RNA samples were used for the preparation of two libraries each, either covering all transcripts or being specifically enriched for primary transcripts by treatment with terminator exonuclease (TEX, Epicentre). Next, equal amounts of RNA samples were poly(A)-tailed with poly(A) polymerase. The 5'-triphosphate structures were removed with tobacco acid pyrophosphatase (TAP). Afterwards, a 5’-RNA adapter was ligated and the first-strand cDNA was synthesized with an oligo(dT)-adapter primer and MMLV reverse transcriptase. A PCR-based amplification step with a high-fidelity DNA polymerase was then used to increase the cDNA concentration to 20-30 ng/µl. A library-specific barcode for multiplexing was included in the 3'-sequencing adapter. For RIP-seq: RIP-seq samples were constructed according to instructions of the NEBnext Multiplex Small RNA library set for Illumina ECL but omitting the TEX treatment.