|
| Status |
Public on Apr 21, 2013 |
| Title |
H3 turnover in clr3 deletion mutant |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
whole cell extract (WCE) genomic DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: clr3 deletion mutant
antibody: None (whole cell extract)
|
| Treatment protocol |
Cells were crosslinked with 1% Formaldehyde
|
| Growth protocol |
Exponentially growing cultures of fission yeast cells expressing an ectopic copy of FLAG tagged histone H3 under the control of inv1 promoter were synchronized by Hydroxyurea and expression of H3-FLAG was induced by changing the carbon source of the medium to Sucrose.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was extracted from cross-linked cells following lysis with bead beater and using micrococcal nuclease (MNase). Chromatin immunoprecipitated DNA recovered with anti-FLAG antibody. After reverse cross-linked and digested with Rnase A and proteinase K, DNA was recovered by Qiagen QIAquick PCR column.
|
| 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 |
clr3 deletion mutant - H3-FLAG IP
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: clr3 deletion mutant
antibody: anti-FLAG
|
| Treatment protocol |
Cells were crosslinked with 1% Formaldehyde
|
| Growth protocol |
Exponentially growing cultures of fission yeast cells expressing an ectopic copy of FLAG tagged histone H3 under the control of inv1 promoter were synchronized by Hydroxyurea and expression of H3-FLAG was induced by changing the carbon source of the medium to Sucrose.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was extracted from cross-linked cells following lysis with bead beater and using micrococcal nuclease (MNase). Chromatin immunoprecipitated DNA recovered with anti-FLAG antibody. After reverse cross-linked and digested with Rnase A and proteinase K, DNA was recovered by Qiagen QIAquick PCR column.
|
| 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
|
| Data processing |
Log ratios of ChIP over WCE were obtained by dividing normalized median Cy5 value over its corrresponding Cy3 value
|
| |
|
| Submission date |
Mar 21, 2013 |
| Last update date |
Apr 21, 2013 |
| 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 |
GPL11244 |
| Series (1) |
| GSE45378 |
HDAC mediated suppression of histone turnover promotes epigenetic stability of heterochromatin |
|
