2 ml lysing matrix e tube

50 ml of E. coli MG1655 was grown to an OD₆₀₀ of 2.0, followed by rapid filtration and immediate freezing in liquid nitrogen. The cells were then resuspended in 1 ml of ice-cold 1× lysis buffer B [20 mM Tris–HCl, pH 7.5, 100 mM NH₄Cl, 10 mM MgCl₂, 1 mM DTT, 1 mM PMSF, 0.4% Triton X 100, 20 U/ml DNase I, 200 U/ml RNase-inhibitor] and lysed using a FastPrep-24 instrument (MP Biomedicals) and a 2 ml lysing matrix E tube (MP Biomedicals) for 15 s at 4 m/s. To remove insoluble debris and the beads, the lysate was cleared by centrifugation for 10 min at 4°C and 16 100 rcf. Of the cleared lysate, 10 μl was mixed with 1 ml TRIzol for the RNA input control. Fifteen A_{260 nm} per ml of the cleared lysate were then layered on top of a linear 10–55% (w/v) sucrose gradient (in 1× lysis buffer B without DNase I or RNase inhibitor and with addition of 5 mM CaCl₂), which was formed in an open-top polyclear ultracentrifugation tube (Seton Scientific) using the Gradient Station model 153. The gradient was centrifuged for 2.5 h at 4°C and 237 000 rcf (35 000 rpm) using an SW 40 Ti rotor, followed by automated fractionation into 20 fractions using an FC 203B fractionator (Gilson). RNA extraction was performed as for the glycerol gradient, except that the vortexing step was performed for 15 s and that DNase treatment of the purified RNA was skipped.