|
| Status |
Public on Oct 22, 2014 |
| Title |
Psc3-GFP_G1_cells-v50_2007 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
input DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: cdc10-v50
antibody: None (whole cell extract)
|
| Treatment protocol |
Exponentially growing cells were cross-linked with paraformadehyde at 18˚C for 30 minutes, followed by dimethyl adipimidate crosslinking at room temperature for 45 min.
|
| Growth protocol |
Wild type and clr4d were cultured in rich media at 30˚C. cdc10-v50 was initially cultured at 26˚C then shifted to 35˚C for 4 hours for G1 arrest.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication and immunoprecipitated by anti-GFP (ab290; Abcam) antibody. Immunoprecipitated DNA was recovered by incubation with protein A slurry and reversed-crosslinked at 65˚C. After reverse cross-linked and digested with Rnase A and proteinase K, DNA was recovered by Qiagen QIAquick PCR column.
|
| 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).
|
| |
|
| Channel 2 |
| Source name |
Psc3 ChIP in cdc10-v50 cells
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/variation: cdc10-v50
chip antibody: anti-GFP (Rb pAb to GFP)
chip antibody vendor: Abcam
chip antibody cat. #: Ab290
chip antibody lot #: GR108426-1
|
| Treatment protocol |
Exponentially growing cells were cross-linked with paraformadehyde at 18˚C for 30 minutes, followed by dimethyl adipimidate crosslinking at room temperature for 45 min.
|
| Growth protocol |
Wild type and clr4d were cultured in rich media at 30˚C. cdc10-v50 was initially cultured at 26˚C then shifted to 35˚C for 4 hours for G1 arrest.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication and immunoprecipitated by anti-GFP (ab290; Abcam) antibody. Immunoprecipitated DNA was recovered by incubation with protein A slurry and reversed-crosslinked at 65˚C. After reverse cross-linked and digested with Rnase A and proteinase K, DNA was recovered by Qiagen QIAquick PCR column.
|
| 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).
|
| |
|
| |
| 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. Background signal was estimated as a median processed signal of 152 oligonucleotides with no homology to S. pombe genome.
Enrichment values were calculated as a log 2 ratio of Cy5 processed signal/ Cy3 processed signal.
|
| |
|
| Submission date |
Apr 16, 2014 |
| Last update date |
Mar 11, 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) |
| GSE56848 |
High resolution Hi-C analysis in S. pombe reveals fundamental elements of genome architecture [ChIP-chip] |
| GSE56849 |
High resolution Hi-C analysis in S. pombe reveals fundamental elements of genome architecture |
|
