|
| Status |
Public on May 19, 2014 |
| Title |
WT_30_r1 |
| Sample type |
SRA |
| |
|
| Source name |
fission yeast cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: wild type
treatment: sorbitol
time (min): 30
replicate: 1
|
| Treatment protocol |
Cells were cultured in yeast extract (YE) medium and harvested immediately before and at 15, 30, 60, 120 and 180 min after addition of 1M sorbitol (Sigma S6021) to the culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the fission yeast cells as follows: cells were treated with hot acidic phenol-chloroform, followed by phenol-chloroform extraction and RNA precipitation. Then RNA was column purified using QIAGEN RNeasy Kit 74104 according to the manufacturers instructions. RNA quality was determined using an Agilent 2100 Bioanalyser. Ribosomal reduction of total RNA (8μg) was performed using the Ribominus Eukaryote Kit for RNA-Seq (Invitrogen A10837-08) and the Ribominus Concentration Module (Invitrogen K155005). Greater than 90% ribosomal reduction was achieved for all samples when assessed on the Agilent 2100 Bioanalyzer.
Libraries were prepared with 350ng of ‘ribosomally-reduced’ total RNA using the SOLiD Total RNA-Seq Kit (Life Technologies 4445374). We used the chemical hydrolysis method to fragment the RNA and performed size selection on Novex 6% TBE-Urea Gels (Invitrogen EC6865). Samples were multiplexed by barcoding with the SOLiD Transcriptome Multiplexing Kit (Life Technologies 4427046). Library quantitation was carried out with the Quant-IT DSDNA HS Assay Kit (Invitrogen Q32851) on the Qubit Fluorometer (Invitrogen Q3287). Molarities were determined using a High Sensitivity DNA Assay (Agilent Technologies 5067-1504) before pooling the libraries in equimolar quantities. Emulsion PCR was carried out on the pooled libraries using an input of 0.5pM in the EZ Bead System to prepare template beads following the manufacturer’s instructions (Life Technologies PN 4448417). Templated beads were then sequenced on a 5500XL Genetic Analyser (Life Technologies 4460730) according to manufacturer’s instructions to generate 50 bases strand-specific reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB 5500xl Genetic Analyzer |
| |
|
| Description |
strand-specific RNA-seq
|
| Data processing |
Sequencing reads were aligned to the fission yeast genome (PomBase database release 11) using SHRiMP2 aligner with default parameters. Total number of reads that can be aligned to the genome at exactly one locus per sample range from 7.5 to 20.1 millions. These uniquely mapped reads were used to identify stretches of unambiguous transcription. Reads that aligned to more than one locus (generally paralogous regions in the genome) were discarded. Adjacent unambiguous transcription regions with minimum peak height of two and located within 50 bases of each other were merged to yield an extensive transcription map of S. pombe. These regions were then positioned relative to known annotation and labelled according to the gene(s) they overlapped with using the Bioconductor package annmap.
The expression of a locus was measured by the Reads Per Kilobase Per Million Mapped (RPKM) score, which was calculated by counting the number of uniquely mapped reads in a given locus and normalizing for by transcript length and for the total read count in the library prep.
Genome_build: PomBase database release 11
Supplementary_files_format_and_content: Tab-delimited text file of RPKM values for all samples
|
| |
|
| Submission date |
Apr 13, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Hui Sun Leong |
| E-mail(s) |
HuiSun.Leong@cruk.manchester.ac.uk
|
| Organization name |
Cancer Research UK Manchester Institute
|
| Department |
The University of Manchester
|
| Lab |
Computational Biology Support Team
|
| Street address |
Wilmslow Road
|
| City |
Manchester |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL18570 |
| Series (1) |
| GSE56761 |
A global non coding RNA system modulates fission yeast protein levels in response to stress |
|
| Relations |
| BioSample |
SAMN02726291 |
| SRA |
SRX516513 |
