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| Status |
Public on Dec 18, 2008 |
| Title |
Isogenic wild-type Rep 1 |
| Sample type |
SRA |
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| Source name |
Isogenic wild-type
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: FY23 x FY86 (Winston et al. 1995), carrying the auxotrophic markers: his3-delta200, leu2-delta1, trp1-delta63, ura3-52.
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| Treatment protocol |
The sequencing libraries were prepared following protocols issued by Illumina (developed by Shujun Luo at Illumina and Brian Williams from Barbara Wold's Lab at CalTech).
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| Growth protocol |
Our unpublished studies suggested that among two dozen or so different conditions that we assayed, exposure to high salt (0.8M NaCl) results in the expression of the greatest fraction of known and novel transcripts, and thus was chosen as the experimental condition to use to find previously unannotated and low abundance transcripts. Cells were grown at 30 degrees C in YPD to approximately 1x10^7 cells/ml as determined by a Beckman Coulter Z2 Particle Count and Size Analyzer. 1.6M NaCl (in YPD) was added in an equal volume of YPD prewarmed to 30 degrees C (final concentration 0.8M). Cells were harvested after 30 minutes by filtration, frozen in liquid nitrogen, and kept at minus 80 degrees C until RNA extraction and purification.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was extracted from the cells using a slightly modified version of the traditional hot phenol protocol (Schmitt et al. 1990) followed by ethanol precipitation and washing. Briefly, 5ml of lysis buffer (10mM EDTA ph 8.0, 0.5% SDS, 10mM Tris-HCl pH 7.5) and 5ml of acid phenol were added to frozen cells and incubated at 60 degrees C for 1 hour with occasional vortexing, then placed on ice. The aqueous phase was extracted after centrifugation and additional phenol extraction steps were performed as needed, followed by a chloroform extraction. Total RNA was precipitated from the final aqueous solution with 10% volume 3M sodium acetate pH 5.2 and ethanol, and resuspended in nuclease-free water. RNA was prepared for sequencing utilizing a protocol obtained from Shujun Luo at Affymetrix and Brian Williams from Barbara Wold's lab at CalTech. Briefly, the total RNA was polyA purified twice using oligo dT conjugated to magnetic beads, which were then fragmented and subjected to cDNA synthesis. Solexa linkers were ligated to the ends of the cDNA, and the cDNA was run on an agarose gel for size selection. An agarose gel slice corresponding to the appropriate size was excised the purified, and the cDNA was amplified via PCR using Solexa specific primers. The PCR product was purified, quantified, and submitted for sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
S. cereviae control RNA
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| Data processing |
The Solexa Pipeline version 0.3.0 produced an output containing our sequences in fastq format. Sequences were matched to both the genome and the ORF sequences using ELAND, tolerating up to 2 mismatches in the first 25 bases.
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| Submission date |
Jun 16, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
Gavin Sherlock |
| E-mail(s) |
sherlock@genome.stanford.edu
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| Phone |
650 498 6012
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| Fax |
650 724 3701
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| URL |
http://genetics.stanford.edu/~sherlock/
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| Organization name |
Stanford University
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| Department |
Genetics
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| Street address |
300 Pasteur Drive
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305-5120 |
| Country |
USA |
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| Platform ID |
GPL9134 |
| Series (2) |
| GSE11801 |
Annotating Low Abundance and Transient RNAs in Yeast using Ultra High-throughput Sequencing |
| GSE11802 |
Annotating Low Abundance and Transient RNAs in Yeast using Tiling Microarrays and Ultra High-throughput Sequencing |
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| Relations |
| SRA |
SRX003157 |
| BioSample |
SAMN02195313 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM298523_080716_GA-EAS46_0001_209DH_L5_eland_extended_pf.txt.gz |
128.9 Mb |
(ftp)(http) |
TXT |
| GSM298523_080716_GA-EAS46_0001_209DH_orf_L5_eland_extended_pf.txt.gz |
121.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available on Series record |
| Raw data are available in SRA |
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