After labeling cells were quickly washed in ice-cold PBS before being scraped in mt-polysome lysis buffer (0.25% Lauryl Maltoside, 10mM Tris pH 7.5, 0.5mM DTT, 20 mM magnesium chloride, 50 mM ammonium chloride 1× EDTA-free protease inhibitor cocktail (Roche)). Cell lysates were dounced 7 times in a 1mL dounce with the tight piston and then flash frozen. 1mL of thawed lysate was clarified by spinning twice at 10,000 rcf. To avoid contamination of nascent RNA still attached to mtDNA into the higher molecular weight sucrose gradient fractions we digested all DNA by 150 units of DNAse I (NEB) in the presence of superaseIn (ThermoFisher) and 0.5mM calcium chloride at room temperature for 1 h. To isolate mitoribosomes, lysates were loaded on 10–50% linear sucrose gradients and centrifuged in a Beckman ultra-centrifuge at 40,000 RPM for 3 h at 4°C using a SW41Ti rotor. Some of the input for the sucrose gradient fractionation was kept and treated as a standard sample described above. Gradients were mixed and fractionated using a BioComp instrument. To identify the mitoribosome containing fractions we western blotted for Mrpl12 (Proteintech 14795–1-AP) and Mrps18B (Proteintech 16139–1-AP) as described below. Monosomes and polysomes containing fractions were pooled and the mitoribosomes were immunoprecipitated out of the pooled fractions. For the immunoprecipitation, MRPL12 antibodies were conjugated to Protein A dynabeads (ThermoFisher) for 1 h in mt-polysome lysis buffer. After washing the beads, lysates were added and incubated for 3 h at 4C. After 3 h the supernatant was removed and the beads were washed three times in mt-polysome lysis buffer before the mitoribosomes were eluted in 0.2% SDS, 100 mM NaCl, 10 mM Tris pH 7.5, 1X EDTA-free protease inhibitor cocktail and SuperaseIn. IP efficiency was confirmed by western blotting as described below. RNA was extracted from the eluates using Trizol LS as described above and then further cleaned by using 1.8x volume of RNAClean XP beads (Beckman Coulter A63987) and washed in 80% ethanol.
When collecting RNA, from NT or FASTKD5 KD cells, destined for direct RNA-sequencing on the nanopore two gradients per condition were used as input for the IP and the final clean-up step using RNAClean XP beads was omitted.