NextSeq 500 sequencing; Mitochondria Ribosome profiling (mitoRiboSeq)  of RF1 Knock out cell line
Mitochondria Ribosome profiling (mitoRiboSeq)  of RF1 Knock out cell line
Medium was discarded and plates were shortly submerged in liquid nitrogen to snap freeze cells. 2x lysis buffer (100 mM Tris pH 7.5, 200 mM NaCl, 40 mM MgCl2, 2 mM DTT, 200 µg/ml chloramphenicol, 200 µg/ml cycloheximide, 2 % Triton X-100, 2x Complete EDTA-free 725 protease inhibitor cocktail (Roche), 4000 U/ml TURBO DNase I (Thermo Fisher)) was added dropwise on the plates and lysates were collected using cell scrapers. Lysis was completed by triturating samples 10 x with a 20-G needle and cell debris were removed by centrifugation (13,000 xg, 20 min, 4 °C). 200 µl of supernatant were subjected to RNase treatment for 30 min at RT (450 U, Ambion RNase I, Thermo Fisher). RNase treatment was stopped by the addition of 10 µl RNase inhibitor (1 U/µl, SUPERase-In, Thermo Fisher), which was followed by a short centrifugation step to remove insoluble material (5,000 xg, 5 min). Supernatants were loaded on 10-30 % sucrose gradients (50 mM Tris pH 7.4, 100 mM NaCl, 20 mM MgCl2, 1 mM DTT, 100 µg/ml chloramphenicol, 100 µg/ml cycloheximide, 1x Complete EDTA-free protease inhibitor cocktail (Roche), 40 U/ml RNase inhibitor (SUPERase-In, Thermo Fisher) in 11 x 34 mm tubes (Beckman Coulter) and run at 39.000 rpm for 2 h 15 min in a TLS-55 rotor (Beckman Coulter). Afterwards, 100 µl fractions were collected, and 10 µl of each were used for western blot analysis. The remaining volumes of fractions 11-16, representing monosome containing fractions, were combined, and further processed for mitoribosome profiling. HEK WT and mtRF1 KO cells were grown to 80 % confluency on 15-cm dishes. RNA was isolated from cells using TRIZOL reagent (Thermo Fisher) according to manufacturer's instructions with the following minor changes: RNA solution was incubated with isopropanol for 30 min, and the pellet was washed with absolute ethanol. RNA was solubilized at 4 °C in water containing 40 units of RNase block (Agilent Technologies). Isolated RNA was heated at 80 °C for 3 min, put on ice  for 1 min, mixed with Gel Loading Buffer II (Thermo Fisher) and loaded onto a 15 % Novex TBE-Urea gel (Thermo Fisher). The gel was run in 1x TBE buffer at 100 V for ~2 h. After completion of the run the gel was stained with 1x SYBR Gold Nucleic Acid Gel Stain (Thermo Fisher) in 1x TBE. Nucleic acids were visualized and bands referring from 30 to 40 nt were excised. RNA was extracted from gel slices in 600 µl RNA extraction buffer (300 mM NaOAc pH 5.5, 1 mM EDTA, 0.25 % SDS) rotating at 4 °C ON. The next day, RNA was precipitated by adding 1.8 ml ice-cold EtOH together with 4 µl GlycoBlue Coprecipitant (Thermo Fisher) and subsequent storage at -80 °C ON. Precipitated RNA was pelleted by centrifugation (5,000 xg, 10 min, 4 °C). Pellet was once washed with 1 ml EtOH, dried for ~5 min and resuspended in 15 µl 10 mM Tris pH 7.5 supplemented with 1 µl RNase inhibitor (SUPERase-In, Thermo Fisher). Samples were heated at 80 °C for 2 min before placing on ice. Next, 3' phosphates were removed by T4 PNK treatment (1 µl T4 PNK (NEB) added) in 1x T4 PNK buffer (NEB) at 37 °C  for 2 h. Reaction was stopped by heat inactivation (65 °C, 10 min). RNA was pelleted by addition of 70 µl water, 2 µl GlycoBlue Coprecipitant (Thermo Fisher), 10 µl 1 M NaOAc and  300 µl EtOH and subsequent storage at -80 °C. RNA was washed and dried as described earlier and finally resuspended in 7 µl 10 mM Tris pH 7.5 supplemented with 1 µl RNase inhibitor. RNA libraries were generated using TrueSeq Small RNA Library Prep Kit (Illumina) according to manufacturer's protocol with some modifications. Preparation was started by  adding 1.2 µl adenylated RA3 to dephosphorylated RNA and incubating the mixture at 80 °C  for 2 min. Afterwards, ligation was performed by addition of 2 µl of T4 RNA Ligase 2 (truncated K227Q), 2 µl T4 RNA Ligase 2 buffer and 6 µl PEG8000 (all components from NEB) and incubation at 14 °C ON. RNA was precipitated as described earlier, 20 µl 3 M NaOAc and 600 µl EtOH) and resuspended in 4 µl 10 mM Tris pH 7.5. Ligation products were then purified on a 15 % Novex TBE-Urea gel (Thermo Fisher), extracted, and precipitated as described earlier. Next, RNA was resuspended in 13 µl 10 mM Tris pH 7.5 supplemented with 1 µl RNase inhibitor. Then, 2 mM ATP, 2 µl 10x T4 PNK buffer and 2 µl T4 PNK (NEB) were added, and the reaction mixture was incubated for 2 h at 37 °C, followed by heat inactivation (65 °C, 10 min). RNA was precipitated and resuspended in 13 µl 10 mM Tris pH 7.5 supplemented with 1 µl RNase inhibitor. Thereafter, RNA footprints were ligated with 5'  RNA adaptor (RA5, Illumina) by adding 1.2 µl RA5, 2 µl 10x T4 buffer and 2 µl T4 RNA ligase 38  (Promega) and incubating at 14 °C ON. RNA was precipitated and resuspended in 3 µl 10 mM Tris pH 7.5. Reverse transcription and PCR amplification of library Reverse transcription was performed using RNA RT primers from TrueSeq Small RNA Library  Prep Kit (Illumina) and SuperScript III First-Strand Synthesis System (Thermo Fisher) according to manufacturer's protocol. Afterwards, 2 µl of RT products were PCR amplified  using Phusion High-Fidelity PCR master mix (NEB) and DNA primers from TrueSeq Small RNA Library Prep Kit (Illumina). The PCR products were purified using QIAquick PCR Purification Kit (Qiagen), precipitated as usual and resolved on a 10 % Novex non-denaturing TBE gel  (Thermo Fisher) using 1x TBE running buffer. PCR products were excised and extracted using DNA extraction buffer (300 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA). Subsequently, PCR products were precipitated and pelleted.