|
| Status |
Public on Dec 31, 2011 |
| Title |
TM_Meiosis_Thiamine |
| Sample type |
RNA |
| |
|
| Source name |
RNA Meiosis 3h + Thiamine
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: h-/h- pat1-114/pat1-114 leu1-32/leu1-32 ade6M210/ade6M216
culture type: Synchronous Meiosis
|
| Treatment protocol |
Minimal Medium (MM) with thiamine (5 microgrames/ml) at 25 ºC (asynchronous) until OD595=0.8 followed by transfer to MM without nitrogen for 14 h after which NH4Cl was added to 0.5 g/l and cells were transferred to 34 ºC to induce synchronous meiosis
|
| Growth protocol |
Minimal Medium (MM) at 25 ºC (asynchronous) until OD595=0.8 followed by transfer to MM without nitrogen for 14 h after which NH4Cl was added to 0.5 g/l and cells were transferred to 34 ºC to induce synchronous meiosis
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared by resuspending the cell pellets in 20 μl extraction buffer (100 mM EDTA, pH 8.0, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0), 20 μl phenol/chloroform, 2 μl 10% SDS, 200 μl glass beads (425-600 μm, SIGMA G-8772). Cells were mechanically disrupted in a Fast-Prep device (Savant BIO 101) and the cell lysate was extracted with phenol, phenol/chloroform and chloroform/isoamyl alcohol before precipitation with 0.3 M sodium acetate and ethanol. RNA was resuspended in 50 μl of sterile water with diethyl pyrocarbonate (SIGMA D-5758) and was further purified with the RNeasy mini kit (Quiagen) following the supplier's specifications.
|
| 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 Meiosis 3h + Thiamine
All the results files are in Wiggle Track Format (WIG) containing: log2(IP fraction/input fraction) for ChIP* samples, log2(mononucleosomal fraction/input fraction) for NUC* samples, and RNA raw signal for TM* samples (transcription mapping). The WIG file coordinates are mapped against the Sanger September 2008 Schizosaccharomyces pombe genome version.
|
| Data processing |
The five M*.CEL were quantile normalized (PMID:12538238). After this, individual raw signals were reported in WIG files.
|
| |
|
| Submission date |
Apr 27, 2011 |
| Last update date |
Dec 31, 2011 |
| 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) |
| GSE28879 |
Nucleosomal organization of replication origins and meiotic recombination hotspots in fission yeast |
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