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| Status |
Public on Mar 15, 2023 |
| Title |
S.flexneri tgt∆, tRNATyr |
| Sample type |
SRA |
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| Source name |
RT via RT-KTq I614Y using 25 µM dCTP
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| Organism |
Shigella flexneri |
| Characteristics |
strain: Shigella flexneri 5a M90T
genotype: tgt{delta}
treatment: growth in full medium (TSB) at 37°C
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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 |
Total RNA was isolated from 50 OD of cells. Cells were harvested and after addition of 1 mL of phenol, glass beads and vigorous shaking for 5 min, samples were centrifuged at 20.000 g for 5 min to clear the cell debris. Equal volume of phenol/chloroform/isoamylalcohol was added to the aqueous phase and centrifuged at 20.000 g for 5 min. After mixing the upper phase with an equal volume of chloroform followed by centrifugation at 20.000 g for 5 min, the RNA was precipitated.
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 |
total RNA
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 |
GPL30040 |
| 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 |
SAMN31535948 |
| SRA |
SRX18088894 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6697806_S.flexneri_Tyr_with_Q.xlsx |
20.2 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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