|
| Status |
Public on May 01, 2009 |
| Title |
Reb1_degron-degron_OFF-culture_B |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
pGAL1::UBR1, pMET3::UBI4::DHFRts::3xHA::REB1, Reb1:dT7 inserted into PRM1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
formaldehyde-crosslinked chromatin digested with MNase to >90% mononucleosomal-sized DNA
pGAL1::UBR1, pMET3::UBI4::DHFRts::3xHA::REB1, Reb1:dT7 inserted into PRM1
|
| Growth protocol |
Culture was grown to log phase (OD=0.7-0.9) in synthetic complete media lacking methionine and cysteine and supplemented with 2% raffinose and 0.1% dextrose at 30 degrees prior to collecting cells
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
culture was crosslinked with 1% formaldehyde for 15 minutes at growth temperature and quenched with 0.125M glycine for 5 minutes at room temperature. Spheroplasts were prepared by digestion with Zymolyase 100T, followed by chromatin digestion with MNase in a buffer containing NP-40 for permeabilization
|
| Label |
Cy3
|
| Label protocol |
DNA was linear amplified by first T-tailing with TdT, then adapting a T7 promoter (using complementary A tails), followed by IVT using a mix of aaUTP:UTP to generate aminoallyl-RNA probe that could be labeled with either Cy3 or Cy5 dye
|
| |
|
| Channel 2 |
| Source name |
reference DNA obtained from strain isogenic for features being probed
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
purified genomic DNA digested with MNase to ~200bp
reference DNA obtained from strain isogenic for features being probed
|
| Growth protocol |
Culture containing reference DNA isogenic to mononucleosome DNA source was grown in rich media to log phase (OD=1.0) prior to harvesting for purification of genomic DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
pure genomic DNA was purified using a Qiagen 100T column as specified by the manufacturer
|
| Label |
Cy5
|
| Label protocol |
DNA was linear amplified by first T-tailing with TdT, then adapting a T7 promoter (using complementary A tails), followed by IVT using a mix of aaUTP:UTP to generate aminoallyl-RNA probe that could be labeled with either Cy3 or Cy5 dye
|
| |
|
| |
| Hybridization protocol |
arrays were blocked with a SDS/BSA solution and each probe was hybridized 1:1 using Agilent hybridization buffer; hybes were allowed to proceed at 65 degrees for at least 12 hours
|
| Scan protocol |
Scanned with a GenePix 4000B at 5 or 10 uM, depending on the size of print tips used to print array
|
| Description |
No further information
|
| Data processing |
Median background subtracted first, then each print block normalized using a loess algorithm
|
| |
|
| Submission date |
Nov 03, 2008 |
| 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 |
GPL7550 |
| Series (1) |
| GSE13446 |
Mechanisms that specify promoter nucleosome location and identity |
|
