GSM1680891: osm RPF; Escherichia coli str. K-12 substr. MC4100; RNA-Seq
To isolate mRNA-bound ribosome complexes we used a previously described approach[Cozzone et al. 1973, J Mol Biol76, 163-179] with some modifications. Exponentially growing cells were poured over crushed ice containing 100 µg/ml chloramphenicol and harvested by centrifugation at 5000xg for 5 min at 4°C. All subsequent operations were carried out on ice with pre-chilled buffers. To obtain polysomal profiles, the cell pellet from 100 ml culture was resuspended in 12 ml of ice-cold sucrose-buffer solution (16 mM Tris pH 8.1 supplemented with 0.5 M RNase-free sucrose, 50 mM KCl, 8.75 mM EDTA, 100 µg/ml chloramphenicol, 1.25 mg/ml lysozyme) and gently stirred for 5 min on ice. 0.3 ml of 1M MgCl2 was added to inhibit lysozyme and the suspension was spun at 6000xg for 10 min at 4°C. The pellet was resuspended in 0.7 ml freshly prepared lysis buffer (10 mM Tris pH 7.8 containing 50 mM NH4Cl, 0.01 M MgCl2, 0.2% triton X-100, 100 µg/ml chloramphenicol and 10 U DNase I (RNase-free, Fermentas)). The lysate was clarified by centrifugation at 10000xg for 10min at 4°C and frozen at -80°C if not analysed immediately. Up to 0.4 ml of clarified lysate was layered onto 15 to 50% (w/v) sucrose gradient (buffered in 10 mM Tris pH 7.8 supplemented with 50 mM NH4Cl, 10 mM MgCl2, 0.2% triton X-100, 100 µg/ml chloramphenicol) and centrifuged for 1.5 h at 35,000 rpm in SW 55Ti rotor (Beckman) at 4°C. The gradient was slowly pumped out from the bottom of the tubes with a flow rate set to 0.38 ml/min and the absorbance at 254 nm (A254) was recorded via a flow-through UV spectrophotometer cell (Pharmacia LKB-UV-M II). For the isolation of RPFs, an aliquot of 100 A260 units of ribosome-bound mRNA fraction (prior to ultracentrifugation in the sucrose gradients, see above) was subjected to nucleolytic digestion with 10 units/µl micrococcal nuclease (Fermentas) for 10 min at room temperature in buffer with pH 9.2 (10 mM Tris pH 11 containing 50 mM NH4Cl, 10 mM MgCl2, 0.2% triton X-100, 100 µg/ml chloramphenicol and 20 mM CaCl2). The monosomal fraction was separated by sucrose density gradient (15-50% w/v). The total RNA was isolated from the monosomes using the hot SDS/phenol method. The micrococcal nuclease also cleaved the rRNAs into fragments with a size similar to the RPFs. The sample was enriched predominantly in one rRNA fragment which was removed by subtractive hybridization at 70 0C using a 5’-biotin-5’-GCCTCGTCATCACGCCTCAGCC-3’ DNA oligonucleotide along with µMACS Streptavidin Kit (Myltenyi Biotec) to remove the biotin-labeled DNA/rRNA hybrids. Total RNA was isolated from ~ 6 ml exponentially growing bacteria with GeneJET™ RNAPurification Kit (Fermentas). Prokaryotic mRNA is a minor fraction of the total cellular RNA, therefore mRNAs were enriched by removal of 16S and 23S rRNAs[He et al. 2010, Nat Methods7, 807-812] with a MICROBExpress™Bacterial mRNA Enrichment Kit (Ambion). 20 µl of the enriched mRNA was mixed with equal volume of 2x alkaline fragmentation solution (2 mM EDTA and 100 mM Na2CO3 pH 9.2) and incubated for 40 min at 95 °C. The reaction was stopped by adding 560 µl 300 mM NaOAc pH 5.5, followed by isopropanol precipitation[Ingolia et al. 2010, Methods Enzymol470, 119-142]. For quantification[Jiang et al. 2011, Genome Res21, 1543-1551], after the random fragmentation the RNA sample was spiked with an external 25-nt RNA standard (5’-AAUGAUAAUUCAAGAAUCAUAACGG-3’) which does not align anywhere in the E. coli genome. Typically, 10 ng of spike-in RNA were added to 25.5 µg total extracted RNA. The optimal time for fragmentation of the mRNA was determined using GAPDH mRNA (0.25 µg; Fermentas) and the spectra were recorded with BioAnalyzer (Agilent RNA 6000 Kit). RNA-size selection and generation of the cDNA libraries was performed as described in [Guo et al. 2010, Nature466, 835-840]
GEO Accession
GSM1680891