|
| Status |
Public on Feb 02, 2024 |
| Title |
H3-FLAG ChIP in WT cells |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
early log. growing cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: Wild type 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).
|
| 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 |
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).
|
| |
|
| Channel 2 |
| Source name |
early log. growing cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: Wild type 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.
|
| 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.
|
| |
|
| 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 |
| |
|
| 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 |
|
