|
| Status |
Public on Mar 24, 2014 |
| Title |
mft1 delta pellet vs mft1 delta supernatant (1 of 2) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
mft1 delta pellet (P18k)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: DLY224
genotype: ura3-1, ade2-1, his3-11,5, trp1-1, leu2-3,112, can1-100, mft1::KAN
|
| Treatment protocol |
Experiments were performed according to previously published methods (Assenholt et al., 2008; Rougemaille et al., 2008)
|
| Growth protocol |
Cells were grown in a YPDA at 25°C to a final OD of 0.8-1.0 and subsequently shifted to 37°C for 15 minutes by the addition of an equal volume of pre-warmed medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extraction using a QIAGEN kit
|
| Label |
Cy5
|
| Label protocol |
DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described (Koszul et al., 2004). We used 0.114 μg of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy5-dCTP.
|
| |
|
| Channel 2 |
| Source name |
mft1 delta supernatant (SN2k)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: DLY224
genotype: ura3-1, ade2-1, his3-11,5, trp1-1, leu2-3,112, can1-100, mft1::KAN
|
| Treatment protocol |
Experiments were performed according to previously published methods (Assenholt et al., 2008; Rougemaille et al., 2008)
|
| Growth protocol |
Cells were grown in a YPDA at 25°C to a final OD of 0.8-1.0 and subsequently shifted to 37°C for 15 minutes by the addition of an equal volume of pre-warmed medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extraction using a QIAGEN kit
|
| Label |
Cy3
|
| Label protocol |
DNA fragments obtained from P18k fractions were labeled by random priming and direct incorporation of fluorescent nucleotides as described in previously published methods (Koszul et al., 2004). We used 1.15 μg of of extracted DNA, 12 μg of random hexamers (Invitrogen) and 50 units of Klenow (Biolabs) in each reaction. The final dNTP concentration was: 0.25 mM dATP, dGTP, dTTP, 0.05 mM dCTP and 0.09 mM Cy3-dCTP.
|
| |
|
| |
| Hybridization protocol |
2µg of labelled cDNA is mixed with one volume of 2X GE buffer (Agilent) and 10% of blocking agent. The hybridization sample volume is dispense on the selected microarray and incubated overnight at 65°C in a hybridization oven (Agilent). The array slides are washed 1 minute in "wash 1 buffer" at room temperature and one minute in "wash 2 buffer" at 37°C. The array slides are then air dryed and ready for scanning
|
| Scan protocol |
Scanned on a genepix 4000B scanner (Axon)
|
| Description |
Biological replicate 1 of 2
|
| Data processing |
Scanning software: GenePix Pro 6.1
Normalization using Goulphar WebTool http://transcriptome.ens.fr/goulphar/
Genepix flags are removed from the analysis. Spots are considered as saturating spot above 60000 and removed. Background is not subtracted from the main signal. Global median normalisation method
|
| |
|
| Submission date |
Oct 14, 2011 |
| Last update date |
Mar 24, 2014 |
| Contact name |
sophie lemoine |
| E-mail(s) |
slemoine@biologie.ens.fr
|
| Organization name |
Ecole Normale Supérieure
|
| Department |
Biologie
|
| Lab |
Plateforme transcriptome
|
| Street address |
46 rue d'Ulm
|
| City |
PARIS |
| ZIP/Postal code |
75005 |
| Country |
France |
| |
|
| Platform ID |
GPL4130 |
| Series (1) |
| GSE32980 |
Using tiling arrays to precisely detect DNA targets of THO in yeast |
|
