|
| Status |
Public on Jul 22, 2011 |
| Title |
Cdc45-IP_alpha_factor_cdc7-4, Replicate 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Cdc45-myc ChIP, anti-Myc antibody, cdc7-4 cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
background/strain: W303
genotype: cdc7-4
cell cycle phase: G1
chip antibody: anti-Myc monoclonal antibody (9E11)
antibody vendor: Abcam
antibody catalog number: ab56
antibody lot number: 935312
|
| Treatment protocol |
Log-phase cells were arrested in alpha-factor for three hours prior to ChIP analysis.
|
| Growth protocol |
Cells were grown in YPD at 25 degrees to log phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were performed as described previously (Aparicio et al., 1997 (PMID 9335335)). 50 ml of culture was collected for each ChIP sample. Half of the resulting cell lysate was used for a Cdc45-13Myc IP using an anti-Myc monoclonal antibody (9E11; Abcam ab56).
|
| Label |
Cy3
|
| Label protocol |
Half of the immunoprecipitated DNA and 5 μg of ChIP input DNA were differentially labeled with 2 nmol of either Cy3-dUTP or Cy5-dUTP (GE Healthcare) using 4 μg random nonomer oligo (IDT) and 0.5 μl 50,000 u/ml Klenowexo- (NEB) in 40 μl. IP and input samples were mixed after labeling and unincorporated dyes were removed using a microcon filter (Millipore YM-30) by washing the samples three times with 0.4 ml TE.
|
| |
|
| Channel 2 |
| Source name |
ChIP input DNA, cdc7-4 cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
background/strain: W303
genotype: cdc7-4
cell cycle phase: G1
chip antibody: none
|
| Treatment protocol |
Log-phase cells were arrested in alpha-factor for three hours prior to ChIP analysis.
|
| Growth protocol |
Cells were grown in YPD at 25 degrees to log phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were performed as described previously (Aparicio et al., 1997 (PMID 9335335)). 50 ml of culture was collected for each ChIP sample. Half of the resulting cell lysate was used for a Cdc45-13Myc IP using an anti-Myc monoclonal antibody (9E11; Abcam ab56).
|
| Label |
Cy5
|
| Label protocol |
Half of the immunoprecipitated DNA and 5 μg of ChIP input DNA were differentially labeled with 2 nmol of either Cy3-dUTP or Cy5-dUTP (GE Healthcare) using 4 μg random nonomer oligo (IDT) and 0.5 μl 50,000 u/ml Klenowexo- (NEB) in 40 μl. IP and input samples were mixed after labeling and unincorporated dyes were removed using a microcon filter (Millipore YM-30) by washing the samples three times with 0.4 ml TE.
|
| |
|
| |
| Hybridization protocol |
Standard Agilent hybridization and wash protocols.
|
| Scan protocol |
Agilent scanner.
|
| Description |
Cdc45-myc cdc7-4.2
Cdc45-myc ChIP in cdc7-4 cells arrested in G1 at 25°C.
|
| Data processing |
For each cohybridization, Cy3 and Cy5 levels were calculated using Agilent Feature Extractor CGH software. Log2 ratios for each experiment and scale normalizations across each set of duplicated experiments were calculated using the sma package (Yang et al. 2002) in R, a computer language and environment for statistical computing (v2.1.0, http://www.r-project.org).
|
| |
|
| Submission date |
May 31, 2011 |
| Last update date |
Jul 23, 2011 |
| Contact name |
Stephen P Bell |
| E-mail(s) |
[email protected]
|
| Phone |
+1-617-253-2054
|
| Organization name |
MIT
|
| Department |
Biology
|
| Street address |
68-630, 77 Massachusetts Ave.
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL5991 |
| Series (1) |
| GSE29646 |
Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. |
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