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| Status |
Public on Sep 19, 2023 |
| Title |
S. pombe_total tRNA_rep2 |
| Sample type |
SRA |
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| Source name |
yeast cell
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: AEP1 (wild type)
cell type: yeast cell
genotype: FY7385; h- leu1-32 ura4-D18 his3-D3
treatment: grown in YES medium at 30°C
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| Growth protocol |
The S. pombe strain AEP1 was grown in YES medium with or without 0.1 μM of queuine at 30°C and 140 rpm. The E. coli strains (including wt, queA∆, queF∆, and tgt∆) were grown in M9 medium at 37°C and 180 rpm. M9 medium for the deletion strains was supplemented with kanamycin (40 µg/ml). Cells were grown to an OD600 of 1.
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| Extracted molecule |
other |
| Extraction protocol |
In vitro synthesis of tRNAs: The plasmid containing tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis sequence, respectively, was linearized with NsiI, and 3 µg of the linear plasmid were used for in vitro transcription by T7 RNA polymerase (Thermo Fisher Scientific) according to the manufacturer’s instructions. After a 7 h incubation at 37°C and subsequent DNase I treatment, the transcription reaction was purified using phenol/ chloroform/ isoamylalcohol extraction followed by gel filtration on Sephadex G50 (GE Healthcare). Extraction of small RNAs from cells: Small RNAs were extracted according to the manufacturer’s protocol (PureLink™ miRNA Isolation Kit (Invitrogen)) with some adjustments. Briefly, cells were suspended in 1 ml Trizol (Ambion). The samples were vortexed for 2 min after adding 0.2 ml chloroform and glass beads, and then were centrifuged at 16 000 rcf, 4°C for 15 min. After adding 215 µl ethanol to the upper phase, the samples were transferred to a spin cartridge and centrifuged at 12 000 rcf for 1 min. 700 µl of ethanol was added to the flow-through, and the samples were transferred to a new spin cartridge followed by centrifugation at 12 000 rcf for 1 min. After washing the cartridge with wash buffer, small RNAs were eluted with 50 µl of DEPC-treated water.
For library preparation, 20 pmol of in vitro-transcribed tRNAAsp were used as input material. To promote ligation of sequencing adapters to tRNA, 16 pmol of custom double-stranded splint adapter (with CGGU overhang complimentary to 3’ termini of tRNAAsp GCCA) were used for the first ligation. The first ligation reaction was performed in a DNA LoBind tube at 25°C for 45 min. It also contained 1 x RNA ligase2 buffer (New England Biolabs), 5% PEG 8000, 2 mM ATP, 6.25 mM MgCl2, 6.25 mM DTT, and 0.5 units/µl T4 RNA ligase 2 (10 000 units/ml) in a total volume of 20 µl. For library preparation of mixtures of in vitro-transcribed tRNAs, 10 pmol of each of the four tRNAs (tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis) and 16 pmol of each of the two splint adapters (splint adapters with CGGU and UGGU overhang) were used in a reaction volume of 40 µl. Library preparation for tRNAs isolated from cells was performed using 40 pmol of deacylated small RNAs and 32 pmol of splint adapter (8 pmol of each of the four splint adapters). Ligation reaction samples were subsequently electrophoresed on a 7 M urea/TBE PAGE gel (8%), and ligation products were excised and purified from the gel. The concentration of gel-purified ligation products was measured with the Qubit RNA HS Assay Kit (Invitrogen). The second ligation of splint-ligated tRNAs to the RNA Adapter (RMX, included in the SQK-RNA002 kit, Oxford Nanopore Technologies) and subsequent library purification were performed following the Nanopore Sequence-Specific Direct RNA sequencing protocol, except that incubation time of the second ligation was increased from 10 min to 30 min to improve ligation efficiency.
Direct RNA nanopore sequencing
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
MinION |
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| Data processing |
Reference curation: The reference for S. pombe tRNAs contains sequences of all unique mature tRNA from GtRNAdb and all unique mitochondrial tRNAs from Ensembl Fungi, and both 5’ and 3’ splint adaptor sequences were added. For E. coli, sequences of all unique mature tRNAs were from GtRNAdb, and splint adaptor sequences were added.
Base calling and alignment: Base calling was done with Guppy (version 6.3.8) in high-accuracy mode. The resulting fastq reads were processed to convert Us to Ts. Sequence alignment was performed on “passed” and “failed” fastq files separately using an optimal local sequence alignment approach (Smith-Waterman) as implemented in parasail. Bam files from “passed” and “failed” reads were then merged and employed for subsequent analysis.
Analysis of base miscalling: JACUSA2 was employed for determination of base miscalling.
Supplementary files format and content: The excel-files include tRNA, position, Kmer, coverage, mismatch rate (Mis_rate), insertion rate (Ins_rate), deletion rate (Del_rate), and mismatch rate of each nucleotides (A_mm, C_mm, G_mm, and T_mm).
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| Submission date |
Jul 07, 2023 |
| Last update date |
Jan 23, 2024 |
| Contact name |
Yu Sun |
| Organization name |
Humboldt-Universität zu Berlin
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| Department |
Institut für Biologie
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| Lab |
Ann E. Ehrenhofer-Murray
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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 |
GPL31123 |
| Series (1) |
| GSE236838 |
Detection of queuosine and queuosine precursors in tRNAs by direct RNA sequencing |
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| Relations |
| BioSample |
SAMN36357685 |
| SRA |
SRX20940966 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7577896_S.pombe_total_tRNA_rep2.xlsx |
689.5 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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