title: GSM2420421: mRNA profiling of wild type Salmonella enterica serovar Thyphimurium - strain SL1344 polysomes; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; RNA-Seq
library_construction_protocol: The frozen pellet of a 50 ml culture was re-suspended and thawed in 1 ml ice-cold lysis buffer for polysome isolation (10 mM MgCl2, 100 mM NH4Cl, 20 mM Tris.HCl pH 8.0, 20 U/ml of RNase-free DNase I (NEB 2U/µl), 1mM chloramphenicol (or 300µg/ml), 20 µl/ml lysozyme (50mg/ml in water) and 100u/ml SUPERase.In™ RNase Inhibitor (Thermo Fisher Scientific, Bremen, Germany)), vortexed and left on ice for 2 min with periodical agitation. Subsequently, the samples were subjected to mechanical disruption by two repetitive cycles of freeze-thawing in liquid nitrogen, added 5mM CaCl2, 30µl 10% DOC and 1 × complete and EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland) and left on ice for 5 min. Lysates were clarified by centrifugation at 16,000 x g for 10 min at 4°C. For the monosome sample, the supernatant was subjected to MNase (Roche diagnostics Belgium) digestion using 600 U MNase (about~ 1000 U per mg of protein). Digestion of polysomes proceeded for 1h at 25°C with gentle agitation at 400 rpm and the reaction was stopped by the addition of 10 mM EGTA. Next, monosomes were recovered by ultracentrifugation over a 1 M sucrose cushion in polysome isolation buffer without RNase-free DNase I and lysozyme, and with 2 mM DTT added using a TLA‐120.2 rotor for 4 hr at 75,000 rpm and 4°C. For the selective purification of monosomes from polysomes (polysome sample), the supernatant was resolved on 10-55% (w/v) sucrose gradients by centrifugation using an SW41 rotor at 35,000 rpm for 2.5 hr at 4°C. The sedimentation profiles were recorded at 260 nm and the gradient fractionated using a BioComp Gradient Master (BioComp) according to the manufacturer's instructions. Polysome-enriched fractions were pooled and subjected to MNase digestion and monosome recovery as described above.Ribosome-protected mRNA footprints with sizes ranging from 26-34 nucleotides were selected and processed as described  previously [23]with some minor adjustments as described
attributes.tag: GEO Accession
attributes.value: GSM2420421