|
| Status |
Public on Mar 21, 2017 |
| Title |
H3-FLAG ChIP in wild-type cells |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
HU blocked cells_immunoprecipitated DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: wild-type
|
| Treatment protocol |
Mid-log phase cells grown in minimal media supplemented with 8% glucose were treated with 15mM HU for 4h. Cells were switched to minimal media supplemented with 4% sucrose and 15mM HU for 2h for induction of H3-FLAG expression. Cells were fixed in 1% formaldehyde at 30˚C.
|
| Growth protocol |
Cells were grown in minimal media at 30˚C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with anti-FLAG M2 agarose (Sigma, A2220). Immunoprecipitated DNA was recovered by incubation with protein A or 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 |
HU blocked cells_whole-cell extract DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotpye: wild-type
|
| Treatment protocol |
Mid-log phase cells grown in minimal media supplemented with 8% glucose were treated with 15mM HU for 4h. Cells were switched to minimal media supplemented with 4% sucrose and 15mM HU for 2h for induction of H3-FLAG expression. Cells were fixed in 1% formaldehyde at 30˚C.
|
| Growth protocol |
Cells were grown in minimal media at 30˚C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with anti-FLAG M2 agarose (Sigma, A2220). Immunoprecipitated DNA was recovered by incubation with protein A or 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_107_Sep09 or ChIP_1200_Jun14 protocol). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization. Background signal was estimated as a median processed signal of 152 oligonucleotides with no homology to S. pombe genome.
|
| |
|
| Submission date |
Oct 18, 2016 |
| Last update date |
Mar 21, 2017 |
| 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 |
GPL8908 |
| Series (2) |
| GSE88901 |
Snf2 Family Protein Fft3 Suppresses Nucleosome Turnover to Promote Epigenetic Inheritance of Heterochromatin and Proper Replication of the Genome [H3-FLAG ChIP] |
| GSE88904 |
Snf2 Family Protein Fft3 Suppresses Nucleosome Turnover to Promote Epigenetic Inheritance of Heterochromatin and Proper Replication of the Genome |
|
