|
| Status |
Public on Jun 09, 2021 |
| Title |
H3K9me2_hht2G13Dclr4CD_REIID |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
log. growing cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: G13D clr4CD
sample type: H3K9me2 immunoprecipitated DNA
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at room temperature. For myc ChIPs, cultures were incubated for 2 h at 18°C prior to fixation (at 18°C) and were subjected to additional crosslinking with 10 mM DMA for 45 min at room temperature.
|
| Growth protocol |
Standard conditions were used to grow cultures at 32˚C, described in detail in methods
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 400-600bp fragments and immunoprecipitated with H3K9me3 antibody (Abcam, ab8898), H3K9me2 antibody (Abcam, ab115159) or myc antibody (Santa Cruz, 9E10). 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 |
sample type: Whole-cell extract DNA
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at room temperature. For myc ChIPs, cultures were incubated for 2 h at 18°C prior to fixation (at 18°C) and were subjected to additional crosslinking with 10 mM DMA for 45 min at room temperature.
|
| Growth protocol |
Standard conditions were used to grow cultures at 32˚C, described in detail in methods
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 400-600bp fragments and immunoprecipitated with H3K9me3 antibody (Abcam, ab8898), H3K9me2 antibody (Abcam, ab115159) or myc antibody (Santa Cruz, 9E10). 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.
|
| Description |
ChIP was performed using anti-H3K9me2 antibody (ab115159) followed by microarray analysis
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software (CHIP-v1_95_May07 or ChIP-v1_10_Apr08 protocol). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization.
|
| |
|
| Submission date |
May 25, 2021 |
| Last update date |
Jun 09, 2021 |
| 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 (1) |
| GSE162701 |
A critical density of histone H3 lysine 9 tri-methylation is required for propagation of heterochromatin and epigenetic inheritance |
|
