Tissue homogenizer

Fibroblasts were homogenized in a TRIzol reagent (Life Technologies) using tissue homogenizer (Bertin Technologies). Total RNA and tRNA extraction, tRNA hydrolysis to ribonucleosides and tRNA modification analysis were performed as previously described [4 (link)].
High-performance liquid chromatography coupled to mass spectrometry was performed using a Luna Omega 1.6 μm, Polar-C18 100 Å column (150 mm × 2.1 mm, Phenomenex, Australia). Mass spectrometry parameters were determined for the ribonucleosides using multiple injections of 0.1–1 ng of purified ncm⁵U, mcm⁵U and mcm⁵s²U nucleosides (a generous gift from Sebastian Leidel, University of Bern, Switzerland) and commercially obtained m⁷G (Santa Cruz Biotechnology). Retention times were determined based on the available compounds (ncm⁵U, mcm⁵U, mcm⁵s²U and m⁷G) and for m¹A based on the previously published results [37 (link)]. Peak assignment and quantification were performed using MultiQuant-v2.1.1 (ABSciex) software. Pseudouridine (Ψ) was used to normalize the data. Statistical analysis was performed using Prism v9.4.0 (GraphPad) software. Number of replicates, the statistical test and statistically significant differences are indicated in the figure legend. Differences between groups were considered statistically significant for p  ≤  0.05.

Total RNA was extracted from approximately 100 mg of brain tissue from P60 mice and homogenized with ceramic beads (Sapphire Bioscience) in a TRIzol reagent (Life Technologies) using tissue homogenizer (Bertin Technologies). Total RNA and tRNA extraction, tRNA hydrolysis to ribonucleosides, and tRNA modification analysis were performed as previously described (Kojic et al, 2018 (link)). HPLC coupled to MS was performed using a Luna Omega 1.6 μm, Polar‐C18 100 Å column (150 mm × 2.1 mm, Phenomenex, Australia). Mass spectrometry parameters were determined for targeted ribonucleosides using multiple injections of 0.1–1 ng of purified ncm⁵U, mcm⁵U and mcm⁵s²U nucleosides (a generous gift from Sebastian Leidel, University of Bern, Switzerland) and commercially obtained m⁷G (Santa Cruz Biotechnology). The obtained retention times for these nucleosides were superimposed with the previously published results (Su et al, 2014 (link)) and the same used to obtain retention times for m¹A and pseudouridine (Ψ). MultiQuant‐v2.1.1 (ABSciex) software was used for peak assignment and quantification. Pseudouridine served as an internal normalization standard. The graphs were prepared in Prism v9.0.1 (GraphPad) software.

Whole murine kidneys and snap-frozen human kidney portions were placed in a tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France) with 1 ml of cell lysis buffer (Invitrogen, Carlsbad, CA). RNA was extracted using a preoptimized RNA purification set (QIAGEN, Hilden, Germany) as per the manufacturer’s instructions. The RNA yield was measured using a spectrophotometer (Thermo Fisher Scientific). Concentrations were standardized throughout each experiment by dilution with Nuclease-Free Water (Life Technologies, Carlsbad, CA). RNA underwent reverse transcription to cDNA as per the manufacturer’s instructions (Applied Biosystems, Foster City, CA; Bio-Rad Laboratories, Hercules, CA). Each sample was analyzed by PCR in triplicate using TaqMan reagents (Thermo Fisher Scientific) as per the manufacturer’s instructions. A parametric unpaired t test was used to test for statistical significance.

Approximately 100 mg of whole-brain tissue was collected from 14.5 dpc embryos and homogenized with ceramic beads (Sapphire Bioscience) using TRIzol reagent (Life Technologies) in tissue homogenizer (Bertin Technologies). Total RNA and tRNA isolation, tRNA hydrolysis, and tRNA modification analysis via high-performance liquid chromatography coupled to mass spectrometry was performed as previously described^{26 (link)}. Mass spectrometer parameters were optimized for targeted ribonucleosides using multiple injections of 0.1–1 ng of commercially obtained m⁷G (MetaGene) and purified ncm⁵U, mcm⁵U, and mcm⁵s²U nucleosides (a generous gift from Sebastian Leidel, University of Bern, Switzerland). The obtained retention times were superimposed with the previously published results^{94 (link)}. An analytical method was developed for the simultaneous analysis of modified ribonucleosides of which six compounds of interest were targeted for quantification (ncm⁵U, mcm⁵U, mcm⁵s²U, Ψ, m¹A, m⁷G, and t⁶A). Pseudouridine (Ψ) served as an internal normalization standard. MultiQuant v2.1.1 (AB Sciex) software was used for peak assignment, quantification, and normalization.