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Sample GSM298530 Query DataSets for GSM298530
Status Public on Dec 18, 2008
Title rrp6 lsm1 pat1 Rep 4
Sample type SRA
 
Source name rrp6 lsm1 pat1
Organism Saccharomyces cerevisiae
Characteristics strain: FY23 x FY86 (Winston et al. 1995), carrying the auxotrophic markers: his3-delta200, leu2-delta1, trp1-delta63, ura3-52. The strain contains deletions in the genes RRP6, LSM1, and PAT1.
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).
Growth protocol Our unpublished studies suggested that among two dozen or so different conditions that we assayed, exposure to high salt (0.8M NaCl) results in the expression of the greatest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.
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer
 
Description S. cerevisiae experimental RNA
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.
 
Submission date Jun 16, 2008
Last update date May 15, 2019
Contact name Gavin Sherlock
E-mail(s) sherlock@genome.stanford.edu
Phone 650 498 6012
Fax 650 724 3701
URL http://genetics.stanford.edu/~sherlock/
Organization name Stanford University
Department Genetics
Street address 300 Pasteur Drive
City Stanford
State/province CA
ZIP/Postal code 94305-5120
Country USA
 
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
Relations
SRA SRX003164
BioSample SAMN02195274

Supplementary file Size Download File type/resource
GSM298530_080703_GA-EAS46_0002_209DG_L8_eland_extended_pf.txt.gz 126.3 Mb (ftp)(http) TXT
GSM298530_080703_GA-EAS46_0002_209DG_orf_L8_eland_extended_pf.txt.gz 115.3 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available on Series record
Raw data are available in SRA

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