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| Status |
Public on Jul 11, 2020 |
| Title |
BY4742_WT_Control_rep2 |
| Sample type |
SRA |
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| Source name |
Yeast cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: WT
strain: BY4742
molecule subtype: mRNA degradation intermediate
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| Treatment protocol |
NO treatment with cycloheximide. Cells were harvested quickly by centrifugation.
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| Growth protocol |
Wild type and mutant cells were grown to an OD600 of 0.6 in YPGR (2% galactose + 0.2% raffinose) medium at 30℃, harvested by centrifugation, then washed and resuspended in the same volume of YEPD (2% glucose) medium containing 1 mM auxin. These cells were grown in the presence of auxin for another 20 minutes at 30℃ to deplete Rbg1 completely.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and DNA was removed by incubation with TURBO Dnase.
50μg of DNA-free total RNA was directly ligated to the rP5_RND oligo at 16℃ overnight. The polyadenylated mRNAs were enriched using Dynabeads (dT)25, followed by fragmentation for 5 minutes at 80℃ in RNA fragmentation buffer. Then the samples were primed with random hexamers and reverse transcribed with Superscript II. Second strand cDNA synthesis was performed using a single PCR cycle. Double-stranded cDNA was purified using HighPrep beads, then was bound to Dynabeads M-280 Streptavidin beads. Bound DNA molecules were subjected to end repair, adenine addition, and adaptor ligation, and a final PCR amplification. 300-500bp regions of the samples was seperated by agarose gel electrophoresis and extracted using single-end, 100bp-read NovaSeq6000 Illumina sequencing.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: 5Pseq
The 3' adaptor sequence was identified and removed using the cutadapt package.
The adaptor-removed reads were deduplicated by bbmap based upon random barcode sequences.
The 5' UMI were trimmed by fastx_trimmer.
Non-coding RNAs were filtered by mapping to annotated S.cerevisiae rRNAs, tRNAs, snRNAs, and snoRNAs using Bowtie1.
Unaligned reads were then mapped to the sacCer3 genome using Bowtie2 with the arguments: --local --D 15 -R2 -N 1 -L 20 -i S,1,0,0.75 -S
Reads with multiple alignments, mapping quality values <30, or those containing soft-clipped bases on the 5' end were excluded.
The 5' ends of reads were extracted and the total numbers of reads per genome location were calculated. Reads per million (RPM) values for each genomic locus were calculated using the number of unique reads.
Genome_build: sacCer3
Supplementary_files_format_and_content: csv files. Each line contains the RPM values at every nucleotide for each gene. 5'UTR and 3'UTR were extract from genome as 100nt long for all genes. First column is gene name, second column is the length of the gene. The rest columns are the RPM values.
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| Submission date |
Jul 10, 2020 |
| Last update date |
Jul 11, 2020 |
| Contact name |
Hong Jin |
| Organization name |
University of Illinois at Urbana-Champaign
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| Department |
Biochemistry
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| Street address |
600 S Mathews Ave
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| City |
Urbana |
| State/province |
Illinois |
| ZIP/Postal code |
61801 |
| Country |
USA |
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| Platform ID |
GPL27812 |
| Series (1) |
| GSE154212 |
Molecular Functions of Conserved Developmentally-Regulated GTP-Binding Protein Drg1 in Translation |
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| Relations |
| BioSample |
SAMN15504431 |
| SRA |
SRX8707825 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4666973_P04_rpm_nt.csv.gz |
2.2 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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