|
| Status |
Public on May 08, 2018 |
| Title |
GFP ChIP for Feb1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
log. growing cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: wildtype
fraction: GFP immunoprecipitated DNA
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at room temperature. For Fep-GFP ChIP, cells were further crosslinked with DMA for 45 min at room temperature.
|
| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA). In ChIP experiments performed at 30°C, cultures were grown at 30˚C to OD600~0.5. In ChIP experiments performed at 18°C, cultures were first grown at 30°C to OD600~0.1-0.2 and then shifted to 18˚C for 3 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with H3K9dime antibody (Abcam, ab115159), or with GFP antibody (Abcam, ab290). Immunoprecipitated DNA was recovered by incubation with protein A/G slurry and reversed-crosslinked at 65˚C.
|
| Label |
Cy5
|
| Label protocol |
ChIP and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (ChIP DNA) or Cy3 (whole-cell extract DNA).
|
| |
|
| Channel 2 |
| Source name |
log. growing cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: smt0
fraction: Whole-cell extract DNA
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at room temperature. For Fep-GFP ChIP, cells were further crosslinked with DMA for 45 min at room temperature.
|
| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA). In ChIP experiments performed at 30°C, cultures were grown at 30˚C to OD600~0.5. In ChIP experiments performed at 18°C, cultures were first grown at 30°C to OD600~0.1-0.2 and then shifted to 18˚C for 3 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with H3K9dime antibody (Abcam, ab115159), or with GFP antibody (Abcam, ab290). Immunoprecipitated DNA was recovered by incubation with protein A/G slurry and reversed-crosslinked at 65˚C.
|
| Label |
Cy3
|
| Label protocol |
ChIP and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (ChIP DNA) or Cy3 (whole-cell extract DNA).
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy5-labeled ChIP DNA and Cy3-labeled whole-cell extract DNA were mixed and combined with human Cot1 DNA, Agilent Blocking Agent and Agilent Hybridization buffer, and hybridized to high-density microarrays in Agilent SureHyb hybridization chamber for 24 hours at 65˚C, 10 rpm. After hybridization, slides were washed according to Agilent protocol.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner.
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software (ChIP_1200_Jun14 protocol). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization.
|
| |
|
| Submission date |
Oct 03, 2017 |
| Last update date |
May 08, 2018 |
| 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 |
GPL6503 |
| Series (2) |
| GSE104528 |
The fission yeast Schizosaccharomyces pombe: H3K9me2 and Fep1-GFP ChIP-chip |
| GSE104547 |
The fission yeast Schizosaccharomyces pombe |
|
