|
Status |
Public on Jun 09, 2021 |
Title |
H3K9me3_nmt1hht2G13D20uM_REIID |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
log. growing cells
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
genotype: G13D sample type: H3K9me3 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-H3K9me3 antibody (ab8898) 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 |
Dec 23, 2020 |
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 |
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