|
| Status |
Public on Mar 16, 2008 |
| Title |
Raf1_1_WT |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Schizosaccharomyces pombe whole cell extract (WCE) genomic DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
Chromatin DNA not enriched for Flag-Raf1
|
| Treatment protocol |
Schizosaccharomyces pombe cells crosslinked with paraformadehyde/dimethyl adipimidate
|
| Growth protocol |
Asynchronous growing fission yeast cells at 30 C and cross-linked with paraformadehyde at 18C for 30 minutes, followed by dimethyl adipimidate crosslinking at room temperature for 45 min.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was extracted from cross-linked cells following lysis with bead beater and sonication. After reverse cross-linked and digested with Rnase A and proteinase K, DNA was recovered by phenol/chloroform extraction and ethanol precipitation
|
| Label |
Cy3
|
| Label protocol |
WCE DNA after amplified by random priming PCR incorporating amino-allyl dUTP was covalently coupled with Cy3 mono NHS ester in 100 mM sodium bicarbonate for 1 hr at room temperature. Labeled DNA was recovered by Qiagen QIAquick PCR column
|
| |
|
| Channel 2 |
| Source name |
Schizosaccharomyces pombe chromatin immunoprecipitated DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
Chromatin DNA enriched for Flag-Raf1
|
| Treatment protocol |
Schizosaccharomyces pombe cells crosslinked with paraformadehyde/dimethyl adipimidate
|
| Growth protocol |
Asynchronous growing fission yeast cells at 30 C and cross-linked with paraformadehyde at 18C for 30 minutes, followed by dimethyl adipimidate crosslinking at room temperature for 45 min.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was extracted from cross-linked cells following lysis with bead beater and sonication. After reverse cross-linked and digested with Rnase A and proteinase K, DNA was recovered by phenol/chloroform extraction and ethanol precipitation
|
| Label |
Cy5
|
| Label protocol |
ChIP DNA after amplified by random priming PCR incorporating amino-allyl dUTP was covalently coupled with Cy5 mono NHS ester in 100 mM sodium bicarbonate for 1 hr at room temperature. Labeled DNA was recovered by Qiagen QIAquick PCR column
|
| |
|
| |
| Hybridization protocol |
500 ng of Cy3 labeled WCE DNA was mixed with 500 ng of Cy5 labeled ChIP DNA plus 50 ul Agilent 10X control targets and 250 ul of 2X Agilent Hybridization buffer
|
| Scan protocol |
Slides were scanned using Agilent scanner and image intensity data were extracted and analyzed with Agilent Feature Extraction software version 8.5
|
| Description |
Schizosaccharomyces pombe cells, Flag-Raf1 ChIP(chromatin immunoprecipitation)-on-chip replicate 2
|
| Data processing |
Log ratios of ChIP over WCE were obtained by dividing normalized median Cy5 value over its corrresponding Cy3 value
|
| |
|
| Submission date |
Feb 19, 2008 |
| Last update date |
Apr 09, 2008 |
| Contact name |
Shiv Grewal |
| Phone |
2407607553
|
| Organization name |
NCI
|
| Department |
LBMB
|
| Lab |
Shiv Grewal
|
| Street address |
NCI bldg 37 Rm 6068 9000 Rockville Pike
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL4682 |
| Series (1) |
| GSE10561 |
Roles of the Clr4 methyltransferase complex in nucleation, spreading and maintenance of heterochromatin |
|
