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| Status |
Public on Nov 01, 2023 |
| Title |
tRNA Neo1.0 circuluar, rep 3 |
| Sample type |
SRA |
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| Source name |
DSy162
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell line: DSy162
cell type: yeast cells
genotype: yABA003
treatment: tRNA neo1.0 circular
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| Treatment protocol |
Yeast transformation was undertaken according to a modified protocol described by Daniel Gietz and Woods (2002). Briefly, yeast strains were inoculated into 10 mL of liquid media and incubated with rotation at 30°C overnight. Overnight cultures were then re-inoculated to OD600 = 0.1 and incubated with rotation to a target OD600 of between 0.5 and 1. Cells were then centrifuged at 2,103 g for 5 minutes, washed with 10 mL sterile ddH2O and centrifuged again. Cell pellets were washed with 10 mL of 0.1 M LiOAc, centrifuged at 2,103 g for 5 minutes. 50 μL of cells were mixed with 5 to 10 μL of transforming DNA, 36 μL of 1M LiOAc, 19 μL ddH2O, 25 μL 10 mg/mL herring sperm carrier DNA and 240 μL of 44% PEG 3350. Transformations were incubated at 30°C for 30 minutes before adding 36 μL of DMSO and heat-shock at 42°C for 15 minutes. Cells were pelleted and incubated in 400 μL of 5 mM CaCl2 at room temperature for 10 minutes and plated onto selective media.
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| Growth protocol |
All yeast strains were derivatives of S288C and grown at 30°C unless otherwise specified. A full list of strains used may be found in Table S1. Yeast cells were grown in either YEP/YPD media (10 g/L yeast extract, 20 g/L peptone and with or without 0.64 g/L tryptophan) or Synthetic Complete (SC) media lacking the indicated amino acids, both supplemented with 2% glucose if not stated otherwise. Standard solid media contained 2% agar and cultures were incubated at 30°C. Liquid cultures were cultivated according to standard procedures in glass tubes on a TC-7 roller drum (New Brunswick).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using Tri-zol extraction.
MSR-seq (PMID 35513407)
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Raw sequences reads were demultiplexed using Je to detect encoded barcode reads. Following this, reads were aligned to curated Sacchromyces tRNA reference genome.
The aligned reads were then used to determine RNA sequence abundance using custom python script. RNA modifications were detected based on aligned reads using samtools sort (http://www.htslib.org/,Li et al., 2009) feature sort the reads in a bam file format. Then IGVtools count (https://software.broadinstitute.org/software/igv/igvtools) count feature was utilized to output a wig files using the following parameters: -z 5 -w 1 -e 250 —bases. The resulting wig files were processed using a custom python script to identify nucleotide mutations as well as coverage of aligned reads.
Supplementary files format and content: tsv files with gene name and abundance per sample
Supplementary files format and content: tsv files with mutation, deletion, coverage for each gene position per sample
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| Submission date |
Sep 18, 2023 |
| Last update date |
Nov 01, 2023 |
| Contact name |
Tao Pan |
| E-mail(s) |
taopan@uchicago.edu
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| Phone |
(773) 702-4179
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| Organization name |
University of Chicago
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| Department |
Biochemistry and Molecular Biology
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| Street address |
929 E. 57th Street
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| City |
Chicago |
| State/province |
Illinois |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL31112 |
| Series (1) |
| GSE243449 |
Design, Construction, and Functional Characterization of a tRNA Neochromosome in Yeast |
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| Relations |
| BioSample |
SAMN37443607 |
| SRA |
SRX21811317 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7787306_MA-16-1-M1-S1toS9_S1_L001_L1_bc6_CATC.sam.tsv.gz |
94.7 Kb |
(ftp)(http) |
TSV |
| GSM7787306_MA-16-1-M1-S1toS9_S1_L001_L1_bc6_CATC.tsv.gz |
698 b |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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