|
| Status |
Public on Aug 22, 2017 |
| Title |
Sap1 ChIP in sap1-1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
sap1-1, Sap1 ChIP DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain/background: 972h
genotype/variation: sap1-1
chip antibody: Sap1
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at room temperature. Cells were further crosslinked with DMA for 45 min at room temperature.
|
| Growth protocol |
WT and sap1-1 cells were initially cultured in rich media (YEA) at 26°C and then shifted to 33°C for 6 hours. Hydroxyurea (HU) was used to arrest wild type and sap1-1 cells at S-phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with GFP antibody (Abcam, ab290), Sap1 antibody (antibody was kindly provided by B. Arcangioli) or with Mcm6 antibody (antibody was kindly provided by H. Masukata). Immunoprecipitated DNA was recovered by incubation with protein A slurry and reversed-crosslinked at 65˚C.
|
| Label |
Cy5
|
| Label protocol |
ChIP and whole-cell extract DNA were amplified by random-primed PCR and conjugated with Cy5 (ChIP DNA) or Cy3 (whole-cell extract DNA).
|
| |
|
| Channel 2 |
| Source name |
sap1-1, whole-cell extract DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain/background: 972h
genotype/variation: sap1-1
fraction: whole-cell extract
chip antibody: none
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at room temperature. Cells were further crosslinked with DMA for 45 min at room temperature.
|
| Growth protocol |
WT and sap1-1 cells were initially cultured in rich media (YEA) at 26°C and then shifted to 33°C for 6 hours. Hydroxyurea (HU) was used to arrest wild type and sap1-1 cells at S-phase.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated with GFP antibody (Abcam, ab290), Sap1 antibody (antibody was kindly provided by B. Arcangioli) or with Mcm6 antibody (antibody was kindly provided by H. Masukata). Immunoprecipitated DNA was recovered by incubation with protein A slurry and reversed-crosslinked at 65˚C.
|
| Label |
Cy3
|
| Label protocol |
ChIP and whole-cell extract DNA were 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 G2600D scanner.
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software. Signal was normalized by combined rank consistency filtering with LOWES intensity normalization.
|
| |
|
| Submission date |
Mar 21, 2017 |
| Last update date |
Aug 22, 2017 |
| 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) |
| GSE96882 |
Shelterin Mediates Genome Reorganization in Response to Replication Stress (ChIP-chip) |
| GSE96883 |
Shelterin Mediates Genome Reorganization in Response to Replication Stress |
|
