Instrument: Illumina HiSeq 3000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: To obtain polysomes from S. aureus 1 ml of night culture was diluted in 250 ml of MHB media and grown till an OD600 of 0.5 at 37°C. ATc induction was done for 10 min. To block translation Cm was added till final concentration 100 μg/ml and culture was incubated for 2 min at 37°C and then quickly chilled with ice cubes. After centrifugation pellets were washed in 2 ml of cold Resuspension Buffer (RB) (20 mM Tris (pH 8.0), 10 mM MgCl2, 5 mM CaCl2, 100 mM NH4Cl, 1 mM Cm). After centrifugation pellets were resuspended in 0.8 ml of cold Lysis Buffer (RB plus 0.1% IGEPAL, 0.4% Triton X-100, 100 U/ml RNase-free DNase I (Roche), 0.5 U/μl SUPERaseIn (Ambion), Protease Inhibitors (Roche)). Suspension was incubated on ice for 5 min and frozen in drops in liquid nitrogen. Drops were milled 5 times with 1-min cycles at 30 Hz in CryoMill MM400 (Retsch) in 10 ml grinding jar with 15 mm grinding ball. For cell extract preparation cell powder was thawed in 50 ml tube and centrifugated at 4000 g, 4°C for 1 min. Supernatant (SN) was clarified at 16000 g, 4°C for 1 min and centrifugated in a new tube at 16000g, 4°C for 10 min. To obtain ribosome footprints 1000 U of S7 Micrococcal Nuclease S7 (Roche) was added per 1 mg of nucleic acids. Samples were incubated for 1 h at 25°C with rotation at 190 rpm. Reaction was quenched by the addition of EGTA to a final concentration 6 mM. 0.5 mg of nucleic acids were loaded to the linear sucrose gradients 10-50% prepared on the Gradient Buffer (20 mM Tris (pH 8.0), 10 mM MgCl2, 100 mM NH4Cl. Tubes were centrifuged at 217000 g (35000 rpm, SW41 Ti rotor (Beckman Coulter)) for 3 h at 4°C. Fractions were collected using a UA/6 detector (ISCO). After centrifugation 750 μl of fractions were collected and incubated with 10 μl of proteinase K (NEB, 800 U/ml) and 10 μl of SDS 10% over night at 4°C. RNAs were isolated with phenol-chloroform libraries were prepared as described (Ingolia et al., 2009; McGlincy and Ingolia, 2017)