|
| Status |
Public on Dec 22, 2020 |
| Title |
Rec12-201-6xHis-2xFLAG ChIP in pat1-114, rad50S, GFP-Pir1-SD cells |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
immunoprecipitated Rc12-DNA linkages
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
growth temperature: cells growing in 34˚C
genotype: pat1-114 rad50S cells expressing GFP-Pir1-SD and Rec12-201-6xHis-2xFLAG
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 30˚C. For GFP and c-MYC ChIPs, fixed cultures were subjected to additional crosslinking with 10 mM DMA for 45 min at room temperature. For Rec12-DNA linkages ChIPs, induced meiotic cells were collected without paraformaldehyde or DMA treatment.
|
| Growth protocol |
For H3K9me2 and Pir1 ChIP, standard conditions were used to produce logarithmically growing cultures in rich media (YEA). Cultures were grown at 30˚C. For Rec12-DNA linkages ChIP, pat1-114 cells were nitrogen starved with EMM-N at 26˚C for 6 h to arrest the cells in G1. The starved cultures were centrifuged and resuspended in EMM medium in 34˚C to induce synchronized meiosis for 4.5 h.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed with glass-beads, DNA sheared by sonication to 400-600bp fragments and immunoprecipitated with H3K9me2 antibody (Abcam, ab1220), c-MYC antibody (Biolegend, 9E10), or GFP antibody (Abcam, ab290). Immunoprecipitated DNA was recovered by incubation with protein A/G slurry and reversed-crosslinked at 65˚C. The Rec12-DNA linkages were recovered by incubation with anti-FLAG-agarose (A2220).
|
| 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 |
Whole-cell extract DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
growth temperature: cells growing in 34˚C
genotype: pat1-114 rad50S cells expressing GFP-Pir1-SD and Rec12-201-6xHis-2xFLAG
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 30˚C. For GFP and c-MYC ChIPs, fixed cultures were subjected to additional crosslinking with 10 mM DMA for 45 min at room temperature. For Rec12-DNA linkages ChIPs, induced meiotic cells were collected without paraformaldehyde or DMA treatment.
|
| Growth protocol |
For H3K9me2 and Pir1 ChIP, standard conditions were used to produce logarithmically growing cultures in rich media (YEA). Cultures were grown at 30˚C. For Rec12-DNA linkages ChIP, pat1-114 cells were nitrogen starved with EMM-N at 26˚C for 6 h to arrest the cells in G1. The starved cultures were centrifuged and resuspended in EMM medium in 34˚C to induce synchronized meiosis for 4.5 h.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed with glass-beads, DNA sheared by sonication to 400-600bp fragments and immunoprecipitated with H3K9me2 antibody (Abcam, ab1220), c-MYC antibody (Biolegend, 9E10), or GFP antibody (Abcam, ab290). Immunoprecipitated DNA was recovered by incubation with protein A/G slurry and reversed-crosslinked at 65˚C. The Rec12-DNA linkages were recovered by incubation with anti-FLAG-agarose (A2220).
|
| 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-FLAG-agarose (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 LOWESS intensity normalization.
|
| |
|
| Submission date |
Aug 12, 2020 |
| Last update date |
Dec 05, 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) |
| GSE142486 |
TOR targets a nuclear RNA processing network to support cell proliferation and control developmental gene expression [ChIP-chip] |
| GSE142488 |
TOR targets an RNA processing network to regulate facultative heterochromatin, developmental gene expression and cell proliferation |
|
