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| Status |
Public on Mar 15, 2023 |
| Title |
Detection of Q-modification in RNAs from S. pombe and S. flexneri via reverse transcription using RT-KTq I614Y (Q-MaP-Seq) |
| Organisms |
Shigella flexneri; Schizosaccharomyces pombe |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
In most eukaryotes and bacteria, queuosine (Q) replaces the guanosine at the wobble position of tRNAs harboring a GUN anticodon. To faithfully detect Q-modification in RNAs from Schizosaccharomyces pombe and Shigella flexneri, Q-MaP-Seq was established and applied to tRNAs from S. pombe WT (AEP1) cells and Shigella flexneri WT cells and tgt∆ cells. Q-modification of in vitro-transcribed RNAs and RNAs isolated from S. pombe and S. flexneri followed by reverse transcription using the RT-active DNA polymerase variant RT-KTq I614Y and sequencing of unmodified compared to modified RNAs allowed identification of Q-sites within tRNAs.
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| Overall design |
in vitro-trasncribed tRNAs Asn and Asp were modified with queuine. Furthermore, RNA from S. pombe cells and S. flexneri cells were Q-modified in vivo. RNA from cells was extracted, and reverse transcribed using the RT-KTq I614Y; tRNAs were amplified and subjected to HTS. HTS reads were analyzed for error RT-signatures in Q-modified compared to unmodified tRNAs.
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| Contributor(s) |
Kaur N, Ehrenhofer-Murray AE |
| Citation(s) |
36971106 |
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| Submission date |
Oct 31, 2022 |
| Last update date |
Jun 15, 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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| Platforms (2) |
| GPL16192 |
Illumina MiSeq (Schizosaccharomyces pombe) |
| GPL30040 |
Illumina MiSeq (Shigella flexneri) |
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| Samples (30)
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| GSM6697780 |
in vitro-transcribed tRNA Asp + Q |
| GSM6697781 |
in vitro-transcribed tRNA Asp, 0% Q |
| GSM6697782 |
in vitro-transcribed tRNA Asp, 2% Q |
| GSM6697783 |
in vitro-transcribed tRNA Asp, 3% Q |
| GSM6697784 |
in vitro-transcribed tRNA Asp, 5% Q |
| GSM6697785 |
in vitro-transcribed tRNA Asp, 10% Q |
| GSM6697786 |
in vitro-transcribed tRNA Asp, 15% Q |
| GSM6697787 |
in vitro-transcribed tRNA Asp, 25% Q |
| GSM6697788 |
in vitro-transcribed tRNA Asp, 50% Q |
| GSM6697789 |
in vitro-transcribed tRNA Asp, 75% Q |
| GSM6697790 |
in vitro-transcribed tRNA Asp, 100% Q |
| GSM6697791 |
AEP1 (WT), unmodified; 100µM dCTP |
| GSM6697792 |
AEP1 (WT), Q-modified; 100µM dCTP |
| GSM6697793 |
AEP1 (WT), unmodified; 25µM dCTP |
| GSM6697794 |
AEP1 (WT), Q-modified; 25µM dCTP |
| GSM6697795 |
AEP1 (WT), unmodified; 12.5µM dCTP |
| GSM6697796 |
AEP1 (WT), Q-modified; 12.5µM dCTP |
| GSM6697797 |
AEP1 (WT), unmodified; 6.25µM dCTP |
| GSM6697798 |
AEP1 (WT), Q-modified; 6.25µM dCTP |
| GSM6697799 |
S.flexneri WT, tRNAAsn |
| GSM6697800 |
S.flexneri tgt∆, tRNAAsn |
| GSM6697801 |
S.flexneri WT, tRNAAsp |
| GSM6697802 |
S.flexneri tgt∆, tRNAAsp |
| GSM6697803 |
S.flexneri WT, tRNAHis |
| GSM6697804 |
S.flexneri tgt∆, tRNAHis |
| GSM6697805 |
S.flexneri WT, tRNATyr |
| GSM6697806 |
S.flexneri tgt∆, tRNATyr |
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| Relations |
| BioProject |
PRJNA896186 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE216921_RAW.tar |
530.0 Kb |
(http)(custom) |
TAR (of XLSX) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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