Computational protocol: Association of RNAs with Bacillus subtilis Hfq

Similar protocols

Protocol publication

[…] Five ml of glucose minimal media with 50 µg/ml tryptophan was inoculated by a single colony of either wild-type 168 or MD145 from a freshly streaked plate and incubated without shaking overnight at 37°C. 50 ml minimal medium was inoculated with 0.5 ml of the overnight culture (OD600 ∼1.5) and incubated shaking at 37°C overnight (approximately 20 hours). These cells were then induced with isopropyl β-D-2-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and incubated shaking for five hours at 37°C. The cultures were transferred to 50 mL conical tubes and centrifuged at 2,900×g for seven minutes; the resulting cell pellet was flash frozen in liquid nitrogen and stored at −80°C. Lysates were prepared using a method reported elsewhere . Briefly, cell pellets were resuspended in lysis buffer (20 mM Tris pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM DTT and 10 mg/ml lyzozyme) at placed on ice for ten minutes. The cell suspension was then flash frozen by liquid nitrogen and thawed for two minutes in a 55°C heat block. Approximately 400 µl of sterile glass beads were added and the suspension was subjected to five rounds of vortexing for 30 seconds followed by 30 seconds on ice (5×). The lysate was then centrifuged at 20,000×g for 30 minutes. The supernatant was then transferred to a fresh tube and incubated with 35 µl of anti-FLAG M2 monoclonal antibody (Sigma) for 30 minutes while rocking at 4°C. Subsequently, 75 µl of protein A sepharose (Sigma), pre-washed in 1 ml of lysis buffer lacking lyzozyme, was added to each tube and rocked for an additional 30 minutes at 4°C. Each sample was then centrifuged for two minutes at 500×g and washed 5× with 500 µl lysis buffer. The mixture was then resuspended in 500 µl wash buffer with 300 mM sodium acetate, mixed with 500 µl of phenol:chloroform:isoamyl alcohol (25∶24:1 pH 7.8) and centrifuged at 20,400×g for ten minutes. The aqueous phase was collected from each separation and placed in a new tube with 1.5 ml of 100% isopropyl alcohol and placed at −20°C overnight. To the organic phase (containing the beads), 1 ml of cold acetone was added for protein precipitation. The precipitated RNA was next centrifuged at 20,400×g for 30 minutes, the supernatant was discarded and the pellet was washed with 1 ml of 70% ethanol followed by centrifugation at 20,400×g for 10 minutes. The supernatant was carefully removed and the pellet was air dried for ten minutes at room temperature followed by re-suspension in 20 µl of sterile distilled water. Roughly 1 µg of RNA for the coIP and mock control samples was used for generation of Illumina-compatible cDNAs. The samples were prepared for sequencing by following the manufacturer’s instructions of the Illumina mRNA sequencing kit, except that the initial polyA-enrichment and final gel purification steps were omitted. Instead of the latter, the cDNA was purified using Qiagen PCR cleanup kit after adaptor ligation. The processed samples were sequenced using an Illumina Genome Analyzer (GAIIx) housed within the DNA Sequencing Core Facility at the UT Southwestern Medical Center. The resulting cDNA sequences were mapped onto the B. subtilis genome (NC_000964.3) using ‘Burrows-Wheeler Aligner’ (BWA) software . Subsequent data processing were done using SAMtools and custom-made Python scripts. Mapped reads were visualized using Integrated Genome Browser (IGB). […]

Pipeline specifications

Software tools BWA, SAMtools
Application Genome data visualization
Organisms Bacillus subtilis