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| Status |
Public on Jun 15, 2022 |
| Title |
A_Thiolutin_S63 |
| Sample type |
SRA |
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| Source name |
WT Thiolutin a
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: CB67
genotype: WT
treatment: G2/M-arrest, thiolutin
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| Treatment protocol |
For transcription inhibition, thiolutin (Abcam, ab143556) was added to cell cultures for the final concentration of 20 µg ml-1 for 30 minutes.
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| Growth protocol |
Cells were cultured in YEP medium (1 % yeast extract, 2 % peptone, 40 μg ml-1 adenine) supplemented with 2 % glucose (YEPD) at 30°C. For arrest in G2/M, benomyl (Sigma, 381586)-containing YEPD medium was added to cells growing logarithmically for a final concentration of 80 µg ml-1. Cell cultures were then incubated for 90 minutes at 30°C achieving complete G2/M-arrest. The Schizosaccharomyces pombe strain (Table S1) used for spike-in normalization in RNA-seq was grown in YES medium (0.5 % yeast extract, 3 % glucose, 150 μg ml-1 adenine, 100 μg ml-1 uracil, 100 μg ml-1 leucine, 100 μg ml-1 lysine, 100 μg ml-1 histidine) at 32°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Forty-five ml of S. cerevisiae cell culture (at an OD of 1.0) was harvested, washed in ice-cold RNase-free water twice, and stored at -80°C. In parallel, 5 ml of logarithmically growing S. pombe WT cells was harvested (at an OD of 0.4) for spike-in normalization, washed in ice-cold RNase-free water twice, and stored at -80°C. A cell pellet from S. cerevisiae, and one from S. pombe were combined in the same tube and allowed to thaw on ice. Thereafter, 1 ml of cold Trizol (Thermo Fisher Scientific, 15596026) and cold glass beads (Sigma, G8772) were added to just about cover the liquid. The tubes were vortexed for 8 minutes, and set on ice afterwards. One ml of cold Trizol was again added and a brief vortex was performed. The liquid was extracted to 1.7 ml Costar tubes. The samples were centrifuged at 12000 g for 10 minutes at room temperature, and supernatants were transferred to new Costar tubes. To the samples, 200 μl of chloroform was added (Sigma, 1024451000), and the tubes were shaken by hand for 15 seconds. The samples were allowed to settle for 2 minutes before being centrifuged at 12000 g for 15 minutes at 4°C. The aqueous phase was collected, and chloroform extraction was performed a second time. Thereafter, 500 μl of 2-propanol (Sigma, 1096341000) was added to the samples. The samples were inverted 7 times, and allowed to precipitate for 10 minutes at room temperature. The precipitates were pelleted at 7500 g for 5 minutes at 4°C, and the pellets were washed once with 1 ml of 70% ethanol, airdried, and resuspended in 25 RNase/DNase-free water. Twenty-five mg of extracted RNA was then treated with 10 units of DNase I (Sigma, 4716728001) for 15 minutes at room temperature. Large ribosomal rRNA molecules were then depleted from the samples using the RiboMinus™ Transcriptome Isolation Kit (Thermo Fisher Scientific, K155003) according to the manufacturer’s instructions.
Samples were then prepared for sequencing using the NEBNext Ultra II Directional RNA Library Prep (NEB, E7760S) according to the manufacturer's protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Data processing |
BAM-formatted map files were generated using STAR version 2.7.3a with default parameters. S. pombe reads for spike-in analysis were mapped by bowtie version 1.2.2. "RNA-seq.S_cerevisiae.xlsx" is the expression matrix.
Genome_build: Saccharomyces Genome Database (SGD), identical with sacCer3
Supplementary_files_format_and_content: *.S_cerevisiae.genes.results: The expression values were estimated by RSEM v1.3.1 with the option "--estimate-rspd --strandedness reverse".
Supplementary_files_format_and_content: RNA-seq.S_cerevisiae.xlsx: Expression matrix.
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| Submission date |
Dec 18, 2021 |
| Last update date |
Jun 15, 2022 |
| Contact name |
Ryuichiro Nakato |
| E-mail(s) |
[email protected]
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| Phone |
+81-3-5841-1471
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| Organization name |
The University of Tokyo
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| Department |
Institute for Quantitative Biosciences
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| Lab |
Laboratory of Computational Genomics
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| Street address |
1-1-1 Yayoi
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| City |
Bunkyo-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
113-0032 |
| Country |
Japan |
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| Platform ID |
GPL31112 |
| Series (2) |
| GSE162194 |
Cohesin-dependent chromosome loop extrusion is limited by transcription and stalled replication forks |
| GSE191207 |
Cohesin-dependent chromosome loop extrusion is limited by transcription and stalled replication forks [RNA-seq] |
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| Relations |
| BioSample |
SAMN24199096 |
| SRA |
SRX13447161 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5741641_67_A_Thiolutin_S63.S_cerevisiae.genes.results.gz |
95.3 Kb |
(ftp)(http) |
RESULTS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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