|
| Status |
Public on May 01, 2009 |
| Title |
Abf1-degron_Reb1_expression_C |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Abf1-degron_Reb1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
pGAL1::UBR1, pMET3::UBI4::DHFRts::3xHA::REB1, pMET3::UB14::abf1(M1R), Reb1:dT7 inserted into PRM1
|
| Growth protocol |
Cells that were growing in synthetic complete media lacking methionine and cysteine at 30 degrees were spiked with 2% galactose for 30 minutes and collected by centrifugation at room temperature, then added to rich (YPA) media supplemented with 2% galactose and prewarmed at 37 degrees. After 5 hours of growth at 37 degrees, cells were collected.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected by 1 minute spins at room temperature and flash frozen in liquid nitrogen. Cell pellets were resuspended in TRIZOL and lysed by 2x2.5 minute bead-beatings. Total RNA was recovered by two chloroform extractions and an isopropanol precipitation.
|
| Label |
Cy3
|
| Label protocol |
Total RNA spiked with RNA from the Agilent Dual Color RNAi spike-in kit was reverse transcribed to cDNA with MMLV reverse transcriptase in a reaction that included dT oligos and aminoallyl-dUTP for dye coupling
|
| |
|
| Channel 2 |
| Source name |
control_without_degron
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
pGAL1::UBR1, Reb1:dT7 inserted into PRM1
|
| Growth protocol |
Cells that were growing in synthetic complete media lacking methionine and cysteine at 30 degrees were spiked with 2% galactose for 30 minutes and collected by centrifugation at room temperature, then added to rich (YPA) media supplemented with 2% galactose and prewarmed at 37 degrees. After 5 hours of growth at 37 degrees, cells were collected.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected by 1 minute spins at room temperature and flash frozen in liquid nitrogen. Cell pellets were resuspended in TRIZOL and lysed by 2x2.5 minute bead-beatings. Total RNA was recovered by two chloroform extractions and an isopropanol precipitation.
|
| Label |
Cy5
|
| Label protocol |
Total RNA spiked with RNA from the Agilent Dual Color RNAi spike-in kit was reverse transcribed to cDNA with MMLV reverse transcriptase in a reaction that included dT oligos and aminoallyl-dUTP for dye coupling
|
| |
|
| |
| Hybridization protocol |
equal amounts of labeled cDNA were combined, heated at 100degs for 5 minutes, iced 5 minutes, then supplemented with 25X Fragmentation buffer (Agilent), 10X blocking agent (Agilent), and 2X GEx Hybridization Buffer HI-RPM (Agilent) prior to appliation to the microarray. Hybridizations were carried out at 65 degrees for 18 hours. Arrays were washed once with room-temperature Gene Expression Wash Buffer 1 and once with Gene Expression Wash Buffer 2 at 37 degrees.
|
| Scan protocol |
Scanned with a GenePix 4000B at 5 uM
|
| Description |
No further information
|
| Data processing |
A loess curve was calculated for the 1:1 DCP spike-in probes, and another loess curve was calculated for the bona fide S. cerevisiae probes. Features were normalized using a composite loess algorithm (Nucleic Acids Res. 30:e15)
|
| |
|
| Submission date |
Jan 07, 2009 |
| Last update date |
Apr 21, 2009 |
| Contact name |
Hiten Madhani |
| E-mail(s) |
hitenmadhani@gmail.com
|
| URL |
http://madhanilab.ucsf.edu
|
| Organization name |
University of California, San Francisco
|
| Department |
Biochemistry and Biophysics
|
| Street address |
600 16th St.
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143-2200 |
| Country |
USA |
| |
|
| Platform ID |
GPL7542 |
| Series (1) |
| GSE13446 |
Mechanisms that specify promoter nucleosome location and identity |
|
