RNA-seq of mouse primary cortical neurons transfected with a synthetic mimic of 5'tiRNA (Gly-GCC) or a scrambled control
RNA-seq of mouse primary cortical neurons transfected with a synthetic mimic of 5'tiRNA (Gly-GCC) or a scrambled control
Total RNA was extracted from mouse primary cortical neurons and spinal cord tissue using Trizol reagent (Invitrogen) according to manufacturer's instructions. Briefly, samples were lysed and homogenised in Trizol reagent and incubated for 5 min at RT. Phase separation was performed by adding chloroform (Sigma-Aldrich) and shaking the samples for 15 s. The samples were incubated for 3 min at RT and then centrifuged at 12000 x g for 15 min at 4°C. The upper aqueous phase was transferred into a new tube and an equal volume of isopropanol (Sigma-Aldrich) was added. Samples were incubated overnight at -20°C and then centrifuged at 12000 x g for 15 min at 4°C. The RNA pellets were washed with 75% ethanol (Sigma-Aldrich), vortexed briefly and then centrifuged at 7500 x g for 10 min at 4°C. The RNA pellets were air-dried for 10 min at RT and then resuspended in 20 μl nuclease-free H2O (Invitrogen) containing 1 μl RNaseOUT RNase inhibitor (Invitrogen). The samples were incubated for 10 min at 65°C, cooled on ice and spun down. The RNA purity and yield was analyzed using a Nanodrop 2000 spectrophotometer (Thermo Scientific).  Cultures of primary cortical neurons were prepared from C57BL/6 mouse embryos at embryonic day 16 (E16) as previously described1. Dissociated cortical neurons were resuspended in plating medium consisting of MEM (Gibco) supplemented with 10% horse serum (Gibco), 10% fetal bovine serum (Sigma-Aldrich), 0.5 mM GlutaMAX (100X, Gibco), 1% Penicillin-Streptomycin (10,000 U/ml, Gibco), 0.6% (w/v) D-glucose (Sigma-Aldrich), 1 mM pyruvic acid (Gibco) and 0.22% (w/v) NaHCO3 (Sigma-Aldrich). Cells were seeded onto poly-L-lysine-coated (0.1 mg/ml, Sigma-Aldrich) 6-well plates at a density of 1.4x106 cells/well and cultured in a controlled humified incubator at 37˚C and with 5% CO2. 4h after plating the cells, the plating medium was replaced with fresh serum-free Neurobasal plus medium (Gibco) supplemented with 0.5 mM GlutaMAX (100X), 10 µg/ml Gentamicin and 1x B27 plus supplement (50x) (Gibco). On DIV 1, cultures were treated with 1 µM cytosine arabinoside (AraC, Sigma-Aldrich) to inhibit growth of non-neuronal cells. Half media changes were performed every three to four days until the cells were used for further experiments.  Transfection protocol A synthetic mimic of endogenous 5'tiRNAGly-GCC and a non-targeting scrambled 5'tiRNA sequence (scrambled control) were synthesised and purified by Integrated DNA Technologies (IDT). The mimics contained a phosphorylated 3'-end and a biotinylated 5'-end and the sequences were as follows: 5'tiRNAGly-GCC, 5'Bio-GCAUUGGUGGUUCAGUGGUAGAAUUCUCGCCUGC-3'Phospho; and scrambled control, 5'Bio-UGUGAGUCACGUGAGGGCAGAAUCUGCUC-3'Phospho. On DIV 8, cultures of mouse primary cortical neurons were transfected with synthetic 5'tiRNAGly-GCC mimic (100 nM) or scrambled control (100 nM) using NeuroMag transfection reagent and protocol (Oz Biosciences). Following transfection, neurons were cultured under standard conditions for 24h until processed for RNA-seq. Total RNA was extracted from mouse primary cortical neurons using Trizol reagent (Invitrogen) according to manufacturer's instructions, and resuspended in 20 μl RNase-free water containing 1 μl RNaseOUT RNase inhibitor (Invitrogen).  Whole transcript RNA-seq was performed by VIB-KU Leuven on total RNA extracted from primary neurons transfected with either 5'tiRNAGly-GCC mimic or scrambled control (n=5 independent experiments) using the KAPA stranded mRNA-seq Kit (Roche) according to manufacturer's instructions. Briefly, sequencing libraries were prepared by converting the RNA to cDNA followed by adapter ligation and enrichment of exon coding sequences by PCR using sequence-specific probes.