GSM6697685: mitoRPF_1_fraction_8_RPF; Homo sapiens; OTHER
The protocol for Mitochondrial ribosome enrichment was adapted from Hsin-Jung Li et al., 2021 (PMID: 33953394). Briefly, 20×106 PBMC cells were washed with PBS containing 100 μg/mL chloramphenicol and 100 μg/mL cycloheximide, pelleted and flash-frozen in liquid nitrogen. The cell pellet was resuspended in 1 mL of lysis buffer (20 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM MgCl2, 100 μg/mL chloramphenicol, 100 μg/mL cycloheximide, 1 mM DTT, 1% (vol/vol) Triton X-100, 0.1% (vol/vol) NP-40, 1× cOmplete protease inhibitor cocktail, 20 U/mL RNasin, 20 U/mL DNase I) and incubated for 10 min on ice. The lysate was homogenized by passing it 3 times through a 26 G needle. Cell debris were removed by centrifugation at 16,000g for 10 min (4 °C) and 100 µL of clarified lysate was kept aside for preparation of RNA-seq libraries. Ribosome protected fragments (RPFs) were generated by nuclease digestion. To this end, 3.75 U/µL of micrococcal nuclease (NEB) and 5 mM CaCl2 were added to the remaining cell lysate; the incubation was performed at room temperature with gentle shaking for 1 h. The reaction was quenched by adding 6 mM of EGTA. The digested lysate was loaded on top of a linear 5-45 % sucrose gradient containing 20 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM MgCl2, 100 μg/mL chloramphenicol, 100 μg/mL cycloheximide and 1 mM DTT and centrifuged at 35,000 rpm, 4 °C for 2 hr using a Beckmann SW40 rotor. The gradient was then fractionated into 20 fractions of 0.57 mL using a Biocomp gradient station fractionator, which allowed recording a UV absorbance profile at 254 nm. Ribosome protected fragments were isolated using Phenol/Chloroform extraction. To that end, 300 µl of the corresponding gradient fractions were mixed with equal amounts of Phenol:Chloroform:IAA (25:24:1, pH 6.6), transferred to a PhaseLock tube (VWR International) and centrifuged at 15,000g for 5 min. A second clean-up step was performed using the isolated aqueous phase and adding equal amounts of Chloroform. After centrifugation, the aqueous phase was isolated and purified using the RNA Clean & Concentrator kit (Zymo Research) by following the manufacturer's instructions for small RNAs. Subsequent library preparation was performed as previously described (PMID: 31959994) with the following modification: ribosomal RNA depletion was skipped for RPF libraries due to low amount of starting material.    For preparation of matched RNA-seq libraries, total RNA was extracted from 40 µL of clarified input lysate using the Direct-zol RNA MiniPrep Kit (Zymo Research). Subsequently, rRNA was depleted using riboPOOLs (siTOOLs Biotech) and following the manufacturer's instructions. Subsequently, RNA-seq libraries were generated as described previously (PMID: 31959994).