characteristics.text: 2025-01-29
characteristics.characteristic: ENA first public
characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: KIS
characteristics.characteristic: INSDC center name
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS19850021
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-14071:Sample 2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: HEK293T
characteristics.characteristic: cell_line
characteristics.text: kidney cell
characteristics.characteristic: cell_type
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Medium was discarded and plates were shortly submerged in liquid nitrogen to snap freeze cells. 300 μl of 2x lysis buffer (100 mM Tris pH 7.5, 200 mM NaCl, 40 mM MgCl2, 2 mM DTT, 200 μg/ml chloramphenicol, 2 % Triton X-100, 2x Complete EDTA-free protease inhibitor cocktail (Roche), 4000 U/ml TURBO DNase I (Thermo Fisher)) was added dropwise on each plate and lysates were collected using cell scrapers. Cell debris were removed by centrifugation (10,000 xg, 15 min, 4 °C). 200 μl of supernatant were subjected to RNase treatment for 30 min at RT (350 U, Ambion RNase I, Thermo Fisher). RNase treatment was stopped by the addition of 15 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, 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. HEK293T WT, TACO1-KO, and TACO1-KO + TACO1 cells were grown to 80 % confluency on 15-cm dishes.  RNA was isolated from pooled fractions using TRIZOL reagent (Thermo Fisher) according to manufacturer’s instructions. 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 (Promega) and incubating at 14 °C ON. RNA was precipitated and resuspended in 3 µl 10 mM Tris pH 7.5.  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 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. Libraries were resuspended in 12 µl 10 mM Tris pH 7.5.  To reduce the amount of ribosomal RNA contamination DSN digestion was performed using a DSN kit (evrogen). First, 4 µl of hybridization buffer (200 mM HEPES pH 7.5, 2 M NaCl) was added to the libraries. Next, libraries were heated for 2 min at 98 °C followed by incubation for 5 h at 68 °C. Consecutively, 1x master buffer (evrogen) together with 2 µl DSN enzyme were added to the samples and incubated additional 25 min at 68°C. Digestion was stopped by addition of 20 µl stop solution (evrogen) and 5 min incubation at 68°C. Finally, samples were cooled down on ice and DNA was isolated by phenol/chloroform extraction. Therefore, samples were mixed with 160 µl water and 200 µl phenol/chloroform (1:1) and the aqueous phase was precipitated as before. 2 µl of digested libraries were subjected to another round of PCR amplification and consecutive gel purification. Final libraries were resuspended in 11 µl 10 mM Tris pH 7.5.
characteristics.characteristic: description
characteristics.text: TACO1 knockout
characteristics.characteristic: genotype
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: Sample 2
characteristics.characteristic: title