|
| Status |
Public on Sep 27, 2014 |
| Title |
Cay1D_repl1 |
| Sample type |
RNA |
| |
|
| Source name |
total RNA extracts of cay1D yeast
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: h- cay1D::kanMX6
|
| Treatment protocol |
untreated
|
| Growth protocol |
Three biological replicates of wildtype and cay1D cells were cultured to exponential phase OD(595)=0.8 in standard yeast extract medium with supplements (YES) at 30°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from yeasts grown to exponential phase using the hot phenol method (Schmitt et al, 1990, Nucleic Acid Research 18: 3091-2) and subjected to 2 DNaseI (QIAGEN) digestion steps. RNA quality was assayed using a 2100 Bioanalyzer (Agilent).
|
| Label |
DNA labeling reagent (Affymetrix)
|
| Label protocol |
Labeled cDNA libraries were constructed according to the GeneChip Eukaryotic Double Strand Whole Transcript Protocol (Affymetrix). In short, total RNA from three biological replicates was reverse transcribed with random primers and SuperScript II (Invitrogen) before performing second strand cDNA synthesis with Klenow fragment (New England Biolabs). Reverse transcription and second strand synthesis were performed in presence of dUTP to allow subsequent fragmentation with Uracil-DNA glycosylase and human apurinic/apyrimidinic endonuclease 1 (Affymetrix). Fragmented double stranded cDNA was labeled with DNA labeling reagent and terminal deoxynucleotidyl transferase.
|
| |
|
| Hybridization protocol |
Samples were hybridized to GeneChip S. pombe Tiling 1.0FR Array containing in total 1,160,624 probes at an average resolution of 20 bp (Affymetrix) according to manufacturer’s instructions.
|
| Scan protocol |
The arrays were scanned using an Affymetrix Scanner 3000 7G according to manufacturer's instruction.
|
| Data processing |
The probes were remapped to the S.pombe genome build from May 2011 (available from ftp://ftp.ebi.ac.uk/pub/databases/pombase/pombe/Archived_directories/GFF/). The impact of the probe-level GC content and overall nucleotide composition on the observed expression values was eliminated using the normalization approaches implemented in the rMAT R package ("PairBinned" method, robust option). After this normalization, the expression values are on a natural log scale.
The result files (.txt) contain the normalized expression values (on a natural log scale) for the individual probes in each of the samples.
|
| |
|
| Submission date |
Sep 26, 2014 |
| Last update date |
Sep 27, 2014 |
| Contact name |
Claus M Azzalin |
| E-mail(s) |
claus.azzalin@bc.biol.ethz.ch
|
| Organization name |
ETH Zürich
|
| Department |
Institute of Biochemistry
|
| Street address |
Otto-Stern-Weg 3
|
| City |
Zürich |
| ZIP/Postal code |
8093 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL7715 |
| Series (1) |
| GSE61792 |
Fission yeast Cactin restricts telomere transcription and elongation by promoting Rap1 pre-mRNA splicing and protein stabilization |
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