|
| Status |
Public on Sep 27, 2011 |
| Title |
Rfa1 ChIP from sub1D cells, replicate 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Rfa1 ChIP
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: sub1 deletion mutant
strain: YF1675
chip antibody: Rfa1
chip antibody purveryor: Rabbit polyclonal antibody was a gift from Steven Brill (Department of Molecular Biology and Biochemistry, Rutgers University). SJ Brill and B Stillman, Genes Dev. 1991 5: 1589-1600.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After immunoprecipitations, DNA was isolated via Pronase digestion and phenol:chloroform extraction, and was amplified by Round A-Round B PCR.
|
| Label |
Cy5
|
| Label protocol |
DNA was Klenow-labeled using Cy3-dCTP or Cy5-dCTP. The Cy5 and Cy3 labelled cDNAs were combined and purified with a Qiagen MinElute ERC cleanup kit.
|
| |
|
| Channel 2 |
| Source name |
input
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: sub1 deletion mutant
strain: YF1675
chip antibody: input
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After immunoprecipitations, DNA was isolated via Pronase digestion and phenol:chloroform extraction, and was amplified by Round A-Round B PCR.
|
| Label |
Cy3
|
| Label protocol |
DNA was Klenow-labeled using Cy3-dCTP or Cy5-dCTP. The Cy5 and Cy3 labelled cDNAs were combined and purified with a Qiagen MinElute ERC cleanup kit.
|
| |
|
| |
| Hybridization protocol |
Amplified, labeled ChIP DNA were mixed, purified by microcon, and hybridized to the microarrays in Agilent hyb buffer for 16 hours at 65 degrees.
|
| Scan protocol |
Fluorescent array images were collected for both Cy3 and Cy5 with a GenePix 4000B fluorescent scanner at 5 micron resolution and image intensity data were extracted and analyzed with GenePix Pro 5.1 analysis software.
|
| Description |
Sample name: TS_3
YF1675 cells (isogenic to BY4741 but bearing a deletion of the Sub1 gene) were grown to an OD of 0.5 in YPDextrose. Cells were crosslinked with formaldehyde (2%), and cell pellets were lysed by bead beating. Chromatin pellets were sonicated, and then subjected to immunoprecipitation with 1.5 ul polyclonal Rfa1 antibody from Steven Brill. As a reference, DNA was obtained from the input chromatin prior to antibody addition.
Raw data file: Agilent11003-3-sub1-rfa1-5f3
|
| Data processing |
After background correction and removal of flagged values, data was block normalized by dilation and log base 2 ratios (Cy5/Cy3) were calculated to produce the values given in the data table.
|
| |
|
| Submission date |
Sep 27, 2011 |
| Last update date |
Sep 27, 2011 |
| Contact name |
Oliver Rando |
| E-mail(s) |
[email protected]
|
| Phone |
508-856-8879
|
| Organization name |
UMass Medical School
|
| Street address |
364 Plantation St.
|
| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605-4321 |
| Country |
USA |
| |
|
| Platform ID |
GPL10930 |
| Series (1) |
| GSE32416 |
Sub1 and RPA associate with RNA Polymerase II at different stages of transcription |
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