Ribosome-Bound mRNA Purification and Sequencing

S. aureus cells from 30 mL culture grown in TSB medium to OD₅₅₀ = 1 were harvested by rapid centrifugation and resuspended in 390 μL ice cold 20 mM Tris lysis buffer pH 8.8, containing 10 mM MgCl₂ x 6 H₂O, 100 mM NH₄Cl, 20 mM Tris (pH 8.0), 0.4% Triton-X-100, 4 U DNase, 0.4 μL Superase-In (Ambion), 1mM chloramphenicol. Cells were disrupted by cell homogenization (FastPrep-24, MP Biomedicals) with 0.5 mL glass beads (diameter 0.1 mm) for 30 s at 6.5 m/s followed by incubation on ice for 5 min. These steps were repeated twice. To remove cell debris, cell lysates were centrifuged and subsequently stored at -80°C and 100 A₂₆₀ units of ribosome-bound mRNA fraction were subjected to nucleolytic digestion with 10 units/μl micrococcal nuclease (Thermofisher) 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 rRNA fragments were depleted using S. aureus riboPOOL rRNA oligo set (siTOOLs, Germany) and the library preparation was performed as previously described [46 (link)].