|
| Status |
Public on Jan 05, 2016 |
| Title |
Mtl1-MYC ChIP in dhp1-2 cells |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Mtl1 ChIP DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: dhp1-2 mutant
growth stage: log. growing cells
chip antibody: c-myc antibody (Covance, MMS-150R)
fraction: Mtl1-MYC immunoprecipitated DNA
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 30˚C. For Red1-MYC, Mtl1-MYC and CFP-Mmi1 ChIP, cells were further crosslinked with DMA for 45 min at 30˚C.
|
| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA). Cultures were first grown at 30˚C to OD600=~0.2 and then shifted to 37˚C for 5 hours.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with H3K9dime antibody (Abcam,ab1220), c-myc antibody (Covance, MMS-150R) or with 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).
|
| |
|
| Channel 2 |
| Source name |
Whole-cell extract DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: dhp1-2 mutant
growth stage: log. growing cells
fraction: Whole-cell extract DNA
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 30˚C. For Red1-MYC, Mtl1-MYC and CFP-Mmi1 ChIP, cells were further crosslinked with DMA for 45 min at 30˚C.
|
| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA). Cultures were first grown at 30˚C to OD600=~0.2 and then shifted to 37˚C for 5 hours.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with H3K9dime antibody (Abcam,ab1220), c-myc antibody (Covance, MMS-150R) or with 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).
|
| |
|
| |
| 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 |
Mtl1-MYC ChIP dhp1-2 V3
|
| 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 |
Nov 05, 2015 |
| Last update date |
Jan 05, 2016 |
| 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) |
| GSE74740 |
A conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing [ChIP-chip] |
| GSE74741 |
A conserved factor Dhp1/Rat1/Xrn2 triggers premature transcription termination and nucleates heterochromatin to promote gene silencing |
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