GSM8009806: MitoIP-Ribo_laminin_retapamulin_90s_2; Homo sapiens; OTHER
MitoIP-Thor-Ribo-Seq: Cells were cultured in 15-cm dishes, washed with ice-cold PBS, and lysed with 1200 µl of hypotonic buffer (10 mM HEPES-KOH pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 100 µg/ml cycloheximide, and 100 µg/ml chloramphenicol). The lysate was incubated with anti-TOM22 antibody-conjugated beads from Mitochondria Isolation Kit, Human (Miltenyi Biotec) for 60 min at 4°C. The subsequent wash and elution steps were performed with wash buffer (10 mM HEPES-KOH pH 7.5, 10 mM KCl, 15 mM MgCl2, 1 mM DTT, 100 µg/ml cycloheximide, and 100 µg/ml chloramphenicol), according to the manufacturer's instructions. The eluted mitochondrial fraction was pelleted by centrifugation at 7,000 × g for 10 min at 4°C and resuspended in modified lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 15 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml cycloheximide, 100 µg/ml chloramphenicol, and 2.5 U/ml TURBO DNase). After clarification by centrifugation at 20,000 × g for 10 min at 4°C, the supernatant was used for library preparation. For the mitoribosome run-off assay, HEK293 cells were pre-treated with 100 µg/ml retapamulin for 30, 60, 90, 120 s, or 30 min and then incubated with 100 µg/ml chloramphenicol and 100 µg/ml cycloheximide to arrest translation elongation. Subsequently, cell lysis and mitochondrial purification were performed as described above, adding 100 µg/ml retapamulin to all the solutions. MitoIP-Thor-Ribo-Seq: preadenylated linkers with the antisense sequence of T7 promoter were ligated and footprints were amplified by in vitro transcription.