| Summary | Nanopore-based approach to sequence native tRNA |
| Key findings | A loss of terminal A in CCA tail under stress conditions. In yeast, identified modification interdependency between Ψ55 and m1A58 and m5U54 |
| Sequencing strategy | ONT direct RNA sequencing |
| Raw data | https://www.ebi.ac.uk/ena/browser/view/PRJEB55684 |
| Software/data repository | https://github.com/novoalab/Nano-tRNAseq; https://github.com/novoalab/nanoRMS |
| Experimental protocol | Snap-frozen yeast pellets were resuspended in 660 µl of TRIzol Reagent (Thermo Fisher Scientific, 15596018) with 340 µl of acid-washed and autoclaved 425–600-µm glass beads (Sigma-Aldrich, G8772). The cells were disrupted by vortexing on top speed for seven cycles of 15 seconds and chilling the samples on ice for 30 seconds between cycles. The samples were then incubated at room temperature for 5 minutes, and 200 µl of chloroform was added. After briefly vortexing the suspension, the samples were incubated for 5 minutes at room temperature and centrifuged at 14,000g for 15 minutes at 4 °C. The upper aqueous phase was transferred to a new tube. To precipitate RNA, 1× volume of molecular-grade isopropanol and 1 µl of GlycoBlue co-precipitant (Thermo Fisher Scientific, AM9515) were added and mixed by inverting and incubated for 10 minutes at room temperature. The samples were centrifuged at 14,000g for 15 minutes at 4 °C, and the pellet was then washed with ice-cold 70% ethanol. The pellet was resuspended in nuclease-free water after air drying for 5 minutes on the benchtop, and the RNA purity was measured using a NanoDrop 1000 spectrophotometer. The samples were treated with Turbo DNase (Thermo Fisher Scientific, AM2238) and subsequently cleaned up using a Zymo RNA Clean and Concentrator-5 kit (Zymo Research, R1016) following the manufacturers’ instructions to retain RNAs ≤200 nt. In brief, 1× volume of RNA Binding Buffer was combined with 1× volume of 100% ethanol. Then, 2× volume of the RNA Binding Buffer and ethanol solution was added to the reaction, transferred to a Zymo-IC column and spun at ≥12,000g at room temperature for 1 minute. Next, 1× volume of 100% ethanol was added to the flow-through, which contains the 17–200-nt fraction, and this was transferred to a new Zymo-IC column and spun at ≥12,000g at room temperature for 1 minute. Then, 400 µl of RNA Prep Buffer was added to the column and spun at ≥12,000g at room temperature for 1 minutes, and then 800 µl of RNA Wash Buffer was added, and the column was spun at >12,000g at room temperature for 2 minutes, transferred to a fresh collection tube and spun for 1 minute. The RNA was eluted in nuclease-free water. RNA concentration was determined using Qubit Fluorometric Quantitation; RNA purity was measured with a NanoDrop 1000 spectrophotometer; and the RNA electropherogram was obtained using Agilent 4200 TapeStation RNA HS ScreenTape Assay. tRNAs purified from S. cerevisiae BY4741 WT and Pus4 KO cultures were resuspended in 10 µl of nuclease-free water and deacylated in 95 µl of 100 mM Tris-HCl (pH 9.0) at 37 °C for 30 minutes. Deacylated tRNAs were recovered using Zymo RNA Clean and Concentrator-5 kit (Zymo Research, R1016), following the manufacturer’s instructions to retain RNAs ≥17 nt but increasing the ethanol concentration to 1.3× after the RNA Prep Buffer step. The tRNA profiles were confirmed using Agilent 4200 TapeStation RNA HS ScreenTape Assay. tRNA libraries were prepared using the SQK-RNA002 kit (ONT) with some protocol alterations as described here. All oligonucleotides used in this study were obtained from Integrated DNA Technologies (IDT). The 5′ RNA splint adapter (/5/rCrCrUrArArGrArGrCrArArGrArArGrArArGrCrCrUrGrGrN) was designed to be complementary to the 3′ NCCA overhang of mature tRNAs, and the 3′ splint RNA:DNA adapter (/5Phos/rGrGrCrUrUrCrUrUrCrUrUrGrCrUrCrUrUrArGrGrArArArArArArArArArAAAA) was designed to be complementary to the rest of the 5′ RNA splint adapter, with a short poly(A) segment for the RTA adapter to anneal to. The 5′ and 3′ splint adapters were prepared at a 1:1 molar ratio in a solution of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl and 1 µl of RNasin Ribonuclease Inhibitor (Promega, N251A), with a final concentration of 50 ng µl−1 and heated to 75 °C for 15 seconds and cooled to 25 °C at a rate of 0.1 °C s−1 to hybridize the adapters. DNA oligos with the same sequence as ONT RTA adapters were ordered from IDT and annealed in the same manner as the 5′ and 3′ splint adapters. Deacylated tRNAs were ligated to the pre-annealed 5′ and 3′ splint adapters at a molar ratio of 1.2:1 (assuming an average tRNA length of 90 nt). The ligation was carried out at room temperature for 2 hours in a total reaction volume of 50 µl with 20% PEG 8000 (NEB, B10048), 1× T4 RNA Ligase 2 Buffer (NEB, B0239S), 4 µl of 6 mg ml−1 recombinant E. coli T4 RNA 2 Ligase (made in-house; see below) and 1 µl of RNasin Ribonuclease Inhibitor (Promega, N251A). A 2× volume of room-temperature-equilibrated AMPure RNAClean XP beads (Beckman Coulter, A63987) was then added to the reaction, pipetting gently up and down, and incubated for 15 minutes at room temperature on a Hula Mixer. The beads were washed with freshly prepared 70% ethanol and left to air dry. The samples were eluted by resuspending the beads in nuclease-free water and incubating them for 10 minutes at room temperature on a Hula Mixer. The RNA concentration was determined using RNA HS Qubit Fluorometric Quantification. Then, 200 ng of 5′ and 3′ ligated tRNAs were ligated to the pre-annealed RTA adapters at a molar ratio of 1:2 (roughly 4.3 pmol tRNAs to 8.6 pmol of RTA adapter). The ligation was carried out at room temperature for 30 minutes in a total reaction volume of 15 µl with 1× Quick Ligation Reaction buffer (NEB, B6058S), 1.5 μl of T4 DNA Ligase (NEB, M0202M, 2,000,000 units per milliliter) and 0.5 µl of RNasin Ribonuclease Inhibitor (Promega, N251A). After ligation, a reverse transcription master mix of 13 µl of nuclease-free water, 2 µl of 10 mM dNTPs (NEB, N0447S), 8 µl of 5× Maxima H Minus Reverse Transcriptase Buffer and 2 µl of Maxima H Minus Reverse Transcriptase (Life Technologies, EP0751) were added directly to the reaction, mixed well by pipetting and incubated at 60 °C for 1 hour, 85 °C for 5 minutes and then brought to 4 °C. The linearized tRNAs were cleaned up using 2× AMPure RNAClean XP beads as described for the ligation reaction. Finally, the ONT RMX sequencing adapters were ligated at room temperature for 30 minutes in a total reaction volume of 40 µl with 1× Quick Ligation Reaction buffer (NEB, B6058S), 3 μl of T4 DNA Ligase (NEB, M0202M, 2,000,000 units per milliliter) and 6 µl of RMX adapters. A 2× volume of AMPure RNAClean XP beads was then added and mixed into the reaction by pipetting gently up and down and incubated for 10 minutes at room temperature on a Hula Mixer. The sample was washed twice with 150 μl of WSB (Wash Buffer), in which the pellet was resuspended by flicking the tube. The sample was eluted in 20 μl of ELB (Elution Buffer) and incubated for 10 minutes at room temperature on a Hula Mixer. The final library was prepared by adding 17.5 μl of nuclease-free water and 37.5 μl of vortexed RRB and kept on ice until loading. The MinION flow cell (FLO-MIN-106) was quality controlled, primed and loaded as per the standard ONT SQK-RNA002 protocol. |
| Analysis protocol | Reads were basecalled using Guppy basecaller version 3.6.1 in high-accuracy (hac) mode. All Us were converted to Ts before mapping. Basecalled reads were mapped using minimap2 version 2.17-r941 with recommended parameters (-ax map-on -k15) or sensitive parameters (-ax map-ont -k5) or BWA version 0.7.17-r1188. For BWA, two modes (MEM and SW) were tested, and several sets of parameters were invoked as follows (ordered from the most stringent to the least stringent settings): (1) bwa mem -W13 -k6 -xont2d; (2) bwa mem -W13 -k6 -xont2d -T20; (3) bwa mem -W13 -k6 -xont2d -T10; (4) bwa mem -W9 -k5 -xont2d -T10; and (5) bwa sw -z10 -a2 -b1 -q2 -r1. Reads mapping to the reverse strand (antisense) were assigned as ‘wrong alignments’. |
| Organism/cell line | S cerevisiae |
| Conditions | Hydrogen peroxide oxidative stress, heat stress, loss of tRNA modification enzymes |
| Approximate experimental time | 1-2 days |
| Starting RNA amount | 250 ng small RNA |
| tRNA expression | ++ |
| Base modifications | ++ |
| Charging status | - |
| tRNA processing and fragmentation | - |
| Citation | https://doi.org/10.1038/s41587-023-01743-6 |
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