Approximately 30e6 cells were treated with chloramphenicol (100μg/ml) for 15 minutes and cycloheximide (100μg/ml) for 5 minutes. Cells were lysed in buffer B (20 mM Tris-HCl, pH 7.8, 100mM KCl, 10mM MgCl2, 1% Triton X-100, 2mM DTT, 100 μg/ml chloramphenicol, 100 μg/ml cycloheximide, 1x Complete protease inhibitor). Lysates were centrifuged at 5000 rpm and the supernatant was treated with 2U/μl of RNase I (Ambion) for 40 minutes at room temperature. Lysates were fractionated on a linear sucrose gradient (7% - 47%) using the SW- 41Ti rotor at 36,000 rpm for 2hrs. Fractions enriched in mito-monosomes and cytosolic monosomes were identified by western blotting, pooled and treated with proteinase K (Roche) in 1% SDS. Released RPFs were purified using Trizol reagent (Invitrogen) following the manufacturer’s instructions. RNA was gel-purified on a denaturing 10% polyacrylamide urea (7 M) gel. A section corresponding to 30-33 nucleotides was excised, eluted and ethanol precipitated. The resulting fragments were 3′-dephosphorylated using T4 polynucleotide kinase (New England Biolabs Inc. Beverly, MA, USA) for 6 h at 37°C in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (100 mM MES-NaOH, pH 5.5, 10 mM MgCl2, 10 mM β-mercaptoethanol, 300 mM NaCl). 3′ adaptor was added with T4 RNA ligase 1 (New England Biolabs Inc. Beverly, MA, USA) for 2.5 h at 37°C. Ligation products were 5′-phosphorylated with T4 polynucleotide kinase for 30 min at 37°C. 5′ adaptor was added with T4 RNA ligase 1 for 18 h at 22°C.
GEO Accession
GSM1446870