|
| Status |
Public on Nov 14, 2013 |
| Title |
Sp_RNA_5FU_t000 |
| Sample type |
RNA |
| |
|
| Source name |
RNA 5FU 0 min
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: 972 h-
culture type: Asynchronous mitosis
time: 0 min
|
| Growth protocol |
YE medium at 30 °C to OD595 = 0.2 (~4 × 106 cells/ml)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was immediately extracted using the MasterPure Yeast RNA Purification Kit (Epicentre, Madison, WI) according to the manufacturer’s recommendations. The purified RNA was immediately frozen in liquid nitrogen and stored at -70 °C. The quality and quantity of total RNA were determined by Nanodrop ND-1000 UV spectroscopy (Thermo Scientific), and RNA integrity was checked using a 2100 Bioanalyzer (Agilent Technologies). We obtained high-quality RNA from all the samples since the RNA Integrity Number (RIN) was greater than 9 in each case.
|
| Label |
biotin
|
| Label protocol |
5.5 mg of the fragmented single-stranded DNA samples resuspended in 45 ml were incubated with a labeling master mix containing DNA Labeling Reagent (5 mM), terminal deoxynucleotidyl transferase (30 U/ul) and 1× Terminal Deoxynucleotidyl Transferase Buffer in a thermal cycler as described in the Affymetrix GeneChip Whole Transcript (WT) Sense Target Assay Labeling Manual.
|
| |
|
| Hybridization protocol |
220 ml 1x hybridization mixture containing 5.5 mg of fragmented and labeled DNA target, 7% DMSO, 1.5, 5, 25 and 100 pM of Eukaryotic Hibridization Controls (bioB, bioC, bioD, cre) respectively, and 50 pM control oligonucleotide B2 were prepared using the Affymetrix GeneChip Hybridization Kit as described in the Affymetrix GeneChip Whole Transcript (WT) Sense Target Assay Labeling Manual. The final DNA target mixture was hybridized to Affymetrix S. pombe Tiling 1.0FR arrays and incubated in a hybridization oven at 45 °C and 60 rpm for 17 h ±1h.
|
| Scan protocol |
Affymetrix standard protocol using Affymetrix Fluidics 450 and Scanner 3000
|
| Description |
RNA 5FU 0 min
|
| Data processing |
The eigth *.CEL files were quantile normalized (PMID:12538238). Mean signal intensities (log2) of biological replicates were reported in WIG files.
All the results files are in Wiggle Track Format (WIG) containing RNA log2 raw signal for RNA samples (transcription mapping). The WIG file coordinates are mapped against the Sanger September 2008 Schizosaccharomyces pombe genome version.
|
| |
|
| Submission date |
May 14, 2013 |
| Last update date |
Nov 14, 2013 |
| Contact name |
Luis Quintales |
| Organization name |
Instituto de Biología Funcional y Genómica (IBFG)
|
| Department |
Universidad de Salamanca / CSIC
|
| Street address |
Calle Zacarías González, 2
|
| City |
Salamanca |
| ZIP/Postal code |
37007 |
| Country |
Spain |
| |
|
| Platform ID |
GPL7715 |
| Series (1) |
| GSE46919 |
Transcriptional response of Schizosaccharomyces pombe to 5-Fluorouracil (5FU) |
|
