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Status |
Public on Feb 02, 2024 |
Title |
H3-FLAG ChIP in clr3D cells |
Sample type |
genomic |
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Channel 1 |
Source name |
early log. growing cells
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
genotype: clr3D cells antibody: H3-FLAG
|
Treatment protocol |
Cells were cross-linked with 1% formaldehyde at room temperature for 20 min. Exponentially growing cells were fixed in 3% paraformaldehyde at 30˚C. DMA was also added to the fixation of cells to be used in Pds5 ChIPs. Prior fixation cells were incubated at 18˚C for 2h (Pds5 ChIPs).
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Growth protocol |
Cells carrying pINV1-H3.2-FLAG plasmid grown in EMM-Leu + 8% glucose media to OD 0.1- 0.15 were synchronized by adding 15 mM HU for 4 h before shifting the cells to EMM-Leu-glucose + 4% sucrose media containing 15mM HU for 2 h to induce the expression of H3-FLAG. Standard conditions were used to produce logarithmically growing cultures in rich media (YEA) . Cultures were grown at 30˚C.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 400-600bp fragments and immunoprecipitated with anti-FLAG M2 afffinity gel (Sigma-Aldrich A2220). Immunoprecipitated DNA was recovered by incubation with protein A/G slurry and reversed-crosslinked at 65˚C. Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with H3K9dime antibody (Abcam, ab1220) or GFP antibody (Abcam, ab290). Immunoprecipitated DNA was recovered by incubation with protein A/G slurry and reversed-crosslinked at 65˚C.
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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). ChIP and whole-cell extract DNA was amplified by random-primed PCR and conjugated with Cy5 (ChIP DNA) or Cy3 (whole-cell extract DNA).
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Channel 2 |
Source name |
early log. growing cells
|
Organism |
Schizosaccharomyces pombe |
Characteristics |
genotype: clr3D cells fraction: Whole-cell extract DNA
|
Treatment protocol |
Cells were cross-linked with 1% formaldehyde at room temperature for 20 min. Exponentially growing cells were fixed in 3% paraformaldehyde at 30˚C. DMA was also added to the fixation of cells to be used in Pds5 ChIPs. Prior fixation cells were incubated at 18˚C for 2h (Pds5 ChIPs).
|
Growth protocol |
Cells carrying pINV1-H3.2-FLAG plasmid grown in EMM-Leu + 8% glucose media to OD 0.1- 0.15 were synchronized by adding 15 mM HU for 4 h before shifting the cells to EMM-Leu-glucose + 4% sucrose media containing 15mM HU for 2 h to induce the expression of H3-FLAG. Standard conditions were used to produce logarithmically growing cultures in rich media (YEA) . Cultures were grown at 30˚C.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 400-600bp fragments and immunoprecipitated with anti-FLAG M2 afffinity gel (Sigma-Aldrich A2220). Immunoprecipitated DNA was recovered by incubation with protein A/G slurry and reversed-crosslinked at 65˚C. Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with H3K9dime antibody (Abcam, ab1220) or 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). 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. 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.
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Scan protocol |
Scanned on an Agilent G2505B scanner. Scanned on an Agilent G2505B scanner.
|
Description |
ChIP was performed using anti-FLAG M2 afffinity gel (Sigma-Aldrich A2220) 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. 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.
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Submission date |
Sep 06, 2023 |
Last update date |
Feb 02, 2024 |
Contact name |
Shiv Grewal |
E-mail(s) |
grewals@mail.nih.gov
|
Phone |
240-760-7553
|
Organization name |
National Institutes of Health
|
Department |
National Cancer Institute
|
Lab |
Laboratory of Biochemistry and Molecular Biology
|
Street address |
9000 Rockville Pike
|
City |
Bethesda |
State/province |
MD |
ZIP/Postal code |
20892 |
Country |
USA |
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Platform ID |
GPL8908 |
Series (2) |
GSE242430 |
Specialized replication of heterochromatin domains ensures self-templated chromatin assembly and epigenetic inheritance [ChIP-chips_H3-FLAG] |
GSE242431 |
Specialized replication of heterochromatin domains ensures self-templated chromatin assembly and epigenetic inheritance |
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