GSM6692725: control mito-monosome longer fragments; Homo sapiens; OTHER
For ribosome profiling of isolated mitochondria, freshly harvested cells were rinsed with ice-cold 1X PBS pH 7.4. All steps were performed at 4° C or on ice roughly following previously published methods (Aibara et al. 2018; Clayton and Shadel 2014). Cells were resuspended in a hypotonic buffer (10 mM HEPES KOH pH 7.5, 10 mM NaCl, and 1.5 mM MgCl2) left to swell for 10 min and lysed using glass dounce homogenizer. Cell lysate was mixed with 1 volume of 2x MS homogenization buffer (20 mM HEPES KOH pH 7.5, 420 mM mannitol, 140 mM sucrose, 2 mM EDTA), cleared by centrifugation at 1,300 x g for 5 min, with supernatant cleared again at 3,000 x g for 5 min. Mitochondria were pelleted by centrifugation at 9,500 x g for 20 min, resuspended in resuspension buffer (20 mM HEPES KOH pH 7.5, 250 mM sucrose, 1 mM EDTA). Sucrose step gradients were prepared in SW32.1 tubes by layering different concentration of sucrose in gradient buffer (20 mM HEPES KOH pH 7.5, 1 mM EDTA): 1.5 ml 60% (w/v) sucrose on the bottom, 4.5 ml 32% (w/v) sucrose in the middle and 3 ml 23% (w/v) on top. The mitochondria suspension was loaded onto the sucrose step gradients and centrifuged using a SW32.1 rotor (Beckman-Coulter) at 22,000 x g for 70 min. Mitochondria were harvested from the interface between the 32- and 60% sucrose layer and flash-frozen in liquid N2. Mitochondria were lysed in 5 volumes of polysome buffer (150 mM NaCl, 20 mM Tris-HCl pH7.4, 5 mM MgCl2, 5 mM DTT, 100 µg/ ml cycloheximide, complete EDTA-free protease inhibitors (Roche) and 40 U/ml RNasin plus (Promega)) supplemented with 1% Triton X-100 and 0.5% Sodium deoxycholate. Lysates were incubated on ice for 10 min and cleared by centrifugation at 10000g, 4° C for 10 min. Ribosome footpritnting and library construction was performed as described earlier (Arpat et al. 2020, McGlisny and Ingolia 2017) with few modifications. Libraries were generated from 24-36 nt (monosome footprints) and 36-69 nt (extended range that includes disome footprints) without ribosomal RNA depletion.