GSM6701633: mtRF1 KO; Homo sapiens; OTHER
Cells were homogenized using a Potter S pestle (Sartorius) in 5 ml of fresh mitochondrial isolation buffer (MIB) containing 310 mM sucrose, 10 mM Tris-HCl and 0.05 % BSA (w/v) by centrifugation at 4,500 g for 1 min at 4C. The homogenate was centrifuged at 1,000 g for 10 min at 4C and the supernatant was centrifuged at 4,500 g for 15 min at 4C to isolate mitochondria. Crude mitochondrial pellets were suspended in MIB supplemented with 1x Complete EDTA-free protease inhibitor cocktail (Roche). Isolated mitochondria at 10 mg/ml were lysed in 10 mM Tris-HCl, pH 7.5, 260 mM sucrose, 100 mM KCl, 20 mM MgCl2 and 2 % digitonin in the presence of 40 U/ml RNase inhibitor and 1x Complete EDTA-free Protease inhibitor cocktail for 20 min. The lysate was centrifuged at 9,200 g for 45 min at 4 C, the clarified lysate was loaded on a continuous 10-30% sucrose gradient (in 10mM Tris-HCl, pH 7.5, 100 mM KCl, 20 mM MgCl2 in the presence of RNAse and protease inhibitors) and centrifuged at 71,000 g in an Optima Beckman Coulter preparative ultracentrifuge. Fractions were collected and one third of each fraction was precipitated with 0.02% sodium deoxycholate and 12% trichloroacetic acid (final concentration), washed twice with acetone, and the entire precipitate was resolved by SDS-PAGE. The fraction corresponding to the translating mitoribosome was isolated and digested with RNase T1 (16.7 U/ml, Ambion) for 30 min at 37C and RNA was extracted from the digested fraction using a Direct-zol RNA Microprep Kit  (Zymo Research) incorporating an on-column RNase-free DNase digestion to remove all DNA. The NEXTFLEX Small RNA-seq Kit v3 (Perkin Elmer) was used to prepare the libraries and they were sequenced on an Illumina NovaSeq.