|
| Status |
Public on Jul 12, 2022 |
| Title |
Clr3-HA-CDx2 in tetR-clr4-ON wt cells |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
log. growing cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: clr3-CDx2 tetR-clr4ON
molecule type: HA immunoprecipitated DNA
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 32˚C and subsequently treated with 10 mM dimethyl adipimidate for 45 min.
|
| Growth protocol |
Standard conditions were used to produce exponentially growing cultures in minimal media (EMM) at 32˚C. Cells were pre-cultured for 20 generations in thiamine-free EMM and grown to mid-log phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads in Biospec Mini-Beadbeater 16, DNA was sheared by sonication to ~ 1,000 bp fragments, cleared by centrifugation at 3,000 g and immunoprecipitated with 2ug of HA antibody (HA.11, Santa Cruz Biotechnology). Immunoprecipitated DNA was recovered by incubation with protein G slurry and the crosslink reversed at 65˚C.Input and immunoprecipitated DNA was treated with RNase A (5ug) and Protease K (40ug) and column purified by Qiaquick PCR purification (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
ChIP and input DNA was random-primed PCR amplified and conjugated with Cy5 (ChIP DNA) or Cy3 (input DNA).
|
| |
|
| Channel 2 |
| Source name |
log. growing cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: clr3-CDx2 tetR-clr4ON
molecule type: Whole-cell extract DNA
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 32˚C and subsequently treated with 10 mM dimethyl adipimidate for 45 min.
|
| Growth protocol |
Standard conditions were used to produce exponentially growing cultures in minimal media (EMM) at 32˚C. Cells were pre-cultured for 20 generations in thiamine-free EMM and grown to mid-log phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads in Biospec Mini-Beadbeater 16, DNA was sheared by sonication to ~ 1,000 bp fragments, cleared by centrifugation at 3,000 g and immunoprecipitated with 2ug of HA antibody (HA.11, Santa Cruz Biotechnology). Immunoprecipitated DNA was recovered by incubation with protein G slurry and the crosslink reversed at 65˚C.Input and immunoprecipitated DNA was treated with RNase A (5ug) and Protease K (40ug) and column purified by Qiaquick PCR purification (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
ChIP and input DNA was random-primed PCR amplified and conjugated with Cy5 (ChIP DNA) or Cy3 (input DNA).
|
| |
|
| |
| Hybridization protocol |
Equal amounts of Cy5-labeled immunoprecipitated DNA and Cy3-labeled input DNA were mixed with human Cot1 DNA, blocking agent and hybridization buffer, and hybridized to Agilent high-density microarrays at 65˚C for 24 hours, 10 rpm. After hybridization, slides were processed according to Agilent protocol.
|
| Scan protocol |
Scanned on an Agilent G2505B scanner.
|
| Description |
Clr3-HA-CDx2 HAChIP V3
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software (ChIP_1200_Jun14). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization.
|
| |
|
| Submission date |
Apr 28, 2022 |
| Last update date |
Oct 11, 2022 |
| 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) |
| GSE184466 |
Histone deacetylation primes chromatin to preserve epigenetic memory for self-propagation of heterochromatin domains |
| GSE201802 |
Histone deacetylation primes chromatin to preserve epigenetic memory for self-propagation of heterochromatin domains [Clr3-CDx2] |
|
