|
| Status |
Public on Mar 05, 2012 |
| Title |
Expression Turnover T=60 Rep 1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Genomic DNA from Rap1 Turnover Strain
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
fraction: Genomic DNA from Rap1 Turnover Strain
growth conditions: Log Phase YPD(1 % Yeast Extract 2% Peptone 2% Dextrose)
|
| Growth protocol |
Rap1 Turnover Strain CLy013 was grown in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
100 ml of vegetative cells (OD600 0.6-0.8) were pelleted and resuspended in 2 ml lysis buffer (10 mM Tris-Cl pH 8.0, 100 mM NaCl, 1 mM EDTA, 1% SDS, 2% Triton X-100). The cells were split into 4 tubes (500 ul each) and an equal volume of phenol:chloroform:isoamyl-alcohol was added to each tube. The cells were then lysed with glass beads. The samples were spun down for 5 minutes at top speed and the aqueous layers was transfered to fresh tubes. Nucleic acids were precipitated with 1 ml of 95% ethanol and resuspended in 200 ul of 1X TE + 30 ug RNAse and incubated at 30 C for 30 minutes. 5 M NaCl was added to a final concentration of 500 mM and the samples were precipitated with isopropanol. Pellets were washed 2X with 70% ethanol and dried at room temperature for 30 minutes. The resulting genomic DNA was resuspended in 10 mM Tris-Cl pH 7.4 and pooled and the concentration was determined with a spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Purified genomic DNA (4 micrograms) was labeled with Klenow (NEB) incorporating amino-allyl dUTP (Sigma) at a ratio of 3:2 with dTTP. Reactive Cy Dye (Amersham) was coupled to the amino-allyl of the resulting DNA fragments in the presence of sodium bicarbonate.
|
| |
|
| Channel 2 |
| Source name |
Rap1 Turnover Strain Time Course T=60 Expression
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
fraction: Total RNA
growth conditions: 100 µm alpha-factor YPR(1 % Yeast Extract 2% Peptone 2% Raffinose) + 2% Galactose
|
| Growth protocol |
Yeast were grown overnight in YPD (Yeast Extract 1%, Peptone 2%, Dextrose 2%) and used to inoculate 800 ml of YPR (Yeast Extract 1%, Peptone 2%, Raffinose 2%) to an OD600 of 0.2 (Genesys 20 Spectrophotometer) in a 4 L Erlenmeyer flask. These cells were grown for approximately 4 hours to an OD600 of 0.4 and subsequently arrested using 5 µM alpha-factor (400 µL of 10 mM, GenScript) until 95% of the yeast cells were unbudded (~3hrs). Cells were then induced by adding 40% galactose directly to the growing yeast to a final galactose concentration of 2%. At this time additional alpha factor was added (400 µL of 10 mM, GenScript). At time points 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240 minutes following galactose induction, 35ml of culture was taken at each time point and added immediately to 37% formaldehyde to a final concentration of 1% for 20 minutes of crosslinking. 13 ml were taken and washed once in sterile ice cold water, pelleted, and frozen in liquid nitrogen for subsequent RNA preparation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from frozen samples using hot acid-phenol as previously described (Ausubel 1997).
|
| Label |
Cy5
|
| Label protocol |
Total RNA (30 micrograms) was reverse-transcribed using SuperScript II Reverse Transcriptase (Invitrogen) incorporating amino-allyl dUTP (Sigma) at a ratio of 3:2 with dTTP. Reactive Cy Dye (Amersham) was coupled to the amino-allyl of the resulting DNA fragments in the presence of sodium bicarbonate.
|
| |
|
| |
| Hybridization protocol |
Samples For Ch1 and Ch2 were competitively hybridized to yeast whole genome PCR based spotted arrays (resolution ~1kb). Hybridizations were carried out overnight at 65 C.
|
| Scan protocol |
Images were acquired using a GenePix 4000B scanner and Genepix software (Axon Instruments)
|
| Data processing |
The normalized median log2 ratios (ch2/ch1) were download from UNC microarray database, dye swapped experiments were inverted
|
| |
|
| Submission date |
Feb 17, 2011 |
| Last update date |
Mar 06, 2012 |
| Contact name |
Jason D Lieb |
| E-mail(s) |
[email protected]
|
| Phone |
919-843-3228
|
| Organization name |
UNC-Chapel Hill
|
| Department |
Biology
|
| Lab |
Jason Lieb
|
| Street address |
408 Fordham Hal, CB#3280
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599-3280 |
| Country |
USA |
| |
|
| Platform ID |
GPL4414 |
| Series (1) |
| GSE27377 |
Rap1 Turnover Galactose Induction Time Course |
|
