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| Status |
Public on Mar 15, 2023 |
| Title |
in vitro-transcribed tRNA Asp, 3% Q |
| Sample type |
SRA |
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| Source name |
mixing of unmodified and Q-modified tRNA-Asp to 75% Q-modification RT via RT-KTq I614Y using 6.25 µM dCTP
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: in-vitro transcribed
treatment: in vitro modification with queuine
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| Growth protocol |
S. pombe strain AEP1 was grown in full medium (YES) with or without 0.1 µM queuine at 30°C. S. flexneri WT and tgt∆ strains were grown in full medium (TSB) at 37°C. Cultures were inoculated at an OD600 of 0.2 and grown to an optical density of 1.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The pJET1 vector carrying the tRNA-Asp and tRNA-Asn sequence, respectively, was linearized with NcoI, and 2.5 µg of the linear vector was used for in vitro transcription using the TranscriptAid T7 High Yield Transcription Kit (ThermoFisher) according to the manufacturer’s instructions. Following an 8 hour incubation at 37 °C with nucleotides and the T7 RNA polymerase and subsequent DNase I treatment, the respective tRNA was purified from the reaction using phenol/chloroform extraction followed by gel filtration with Sephadex G50 (GE Healthcare). For in vitro modification of tRNA with queuine, 10 µM of in vitro transcribed tRNA was incubated with 200 nM hTGT (QTRT1:QTRT2) and 5 µM queuine in reaction buffer (50 mM Tris-HCl pH 7.5, 20 mM NaCl, 5 mM MgCl2 and 2 mM dithiothreitol) for 5 h at 37 °C. The RNA was purified using phenol/chloroform extraction and precipitated with 1/10 volume of ammonium acetate and three volumes of 100% ethanol.
Extraction of the RNA was performed as described in the "SAMPLEs" section. 500 ng RNA was hybridized to 500 nM of the respective stemloop-primer with the following thermocycling program: 95 °C for 2 min, 65 °C for 30 sec, ramp down to 4 °C with 5 °C/30 sec. After hybridization and incubation on ice for 1 min, 100 µM of each dNTP and 100 nM RT-KTq I614 DNA polymerase were added. The reverse transcription was carried out in reaction buffer (50 mM Tris-HCl (pH 9.2), 16 mM (NH4)2SO4, 25 mM MgCl2, 0.1% Tween 20) in a total volume of 20 µL. For experiments using decreased dCTP concentrations, 25 µM, 12.5 µM and 6.25 µM dCTP were added to the RT reaction. Following incubation at 55 °C for 1 h and incubation on ice for 10 min, the cDNA was amplified using Taq polymerase. Amplification was performed with 5 µL cDNA in reaction buffer containing 2.5 µM dNTPs, 2 mM MgCl2, 1 x Taq buffer+ KCl and 1.25 U Taq polymerase (Thermo Fisher) in a total volume of 200 µL. Primers binding at the 3’ end contained a barcoded sequence for multiplexing the PCR products. Samples were separated on a 2% agarose gel, and the respective PCR products were gel-purified using the QIAquick Gel Extraktion Kit (Qiagen) according to the manufacturer’s protocol.
Library preparation of amplified tRNAs for deep sequencing was performed using the NEXTflexR qRNA-SeqTM Kit v2 – Set C (Bioo Scientific) according to the manufacturer's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
in vitro-transcribed tRNA
column in processed data file: mm_ratio
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| Data processing |
Adapter-trimming was performed using Skewer version 0.2.2 and trimming of PCR primers, selection of high-quality reads and sorting of the reads based on the sequence in the degenerate region of the RT-primerwas performed using Bioconductor package ShortRead. Reads were trimmed to the expected size of the transcript using Cutadapt version 3.7.
Alignment to RNA sequences from S. pombe (https://www.pombase.org/) and Shigella flexneri (http://www.ensembl.org/) was achieved using Samtools version 1.12 and Bowtie version 2.2.5. Settings were chosen to allow up to one mismatch in seed with a seed length of 6.
Misincorporation was determined using CoverageAnalyzer.
Assembly: S.pombe genome version ASM294v2.29
Supplementary files format and content: excel-files include position, refbase, coverage, total mismatch ratio (mm_ratio), mismatch ratio of single nucleotides (a_mism, g_mism, t_mism and c_mism) and arrest_ratio.
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| Submission date |
Oct 31, 2022 |
| Last update date |
Mar 18, 2023 |
| Contact name |
Ann E. Ehrenhofer-Murray |
| Organization name |
Humboldt-Universität zu Berlin
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| Department |
Institut für Biologie
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| Street address |
Philippstr. 13
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| City |
Berlin |
| ZIP/Postal code |
10099 |
| Country |
Germany |
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| Platform ID |
GPL16192 |
| Series (1) |
| GSE216921 |
Detection of Q-modification in RNAs from S. pombe and S. flexneri via reverse transcription using RT-KTq I614Y (Q-MaP-Seq) |
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| Relations |
| BioSample |
SAMN31535971 |
| SRA |
SRX18088887 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6697783_in_vitro_Asp_3pct_Q.xlsx |
21.0 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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