|
| Status |
Public on Jul 01, 2014 |
| Title |
H2A.Z ChIP in wild-type |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
M2 FLAG immunoprecipitated DNA
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
cell type: pht1-3xflag cells
growth stage: exponentially growing cells
ip method: M2 FLAG slurry (Sigma)
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 30˚C; for Xap5 immunoprecipitation paraformaldehyde fixation was supplemented with DMA (10mM, 45 min, 30˚C) crosslinking.
|
| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA) at 30˚C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated by incubation with anti HA affinity matrix (Roche 3F10) or M2 FLAG slurry (Sigma). Recovered DNA was 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 |
cell type: pht1-3xflag cells
growth stage: exponentially growing cells
|
| Treatment protocol |
Exponentially growing cells were fixed in 3% paraformaldehyde at 30˚C; for Xap5 immunoprecipitation paraformaldehyde fixation was supplemented with DMA (10mM, 45 min, 30˚C) crosslinking.
|
| Growth protocol |
Standard conditions were used to produce logarithmically growing cultures in rich media (YEA) at 30˚C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed with glass-beads, DNA sheared by sonication to 500-1000bp fragments and immunoprecipitated by incubation with anti HA affinity matrix (Roche 3F10) or M2 FLAG slurry (Sigma). Recovered DNA was 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 |
H2A.Z ChIP wt V3
|
| Data processing |
Data were extracted using Agilent Feature Extraction Software (CHIP-v1_95_May07 or ChIP-1100_Jul11 protocol). Signal was normalized by combined rank consistency filtering with LOWES intensity normalization. The enrichment ratios ('enrichment_ratios.txt') were calculated by dividing the normalized ChIP signals by whole cell extract (control) and provided as Series supplementary file.
|
| |
|
| Submission date |
Apr 30, 2013 |
| Last update date |
Jul 01, 2014 |
| Contact name |
Shajahan Anver |
| E-mail(s) |
sanver@ucdavis.edu
|
| Organization name |
University of California Davis
|
| Department |
Plant Biology
|
| Lab |
Harmer Lab
|
| Street address |
1002 LSA, One Shields Av
|
| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
| |
|
| Platform ID |
GPL6503 |
| Series (2) |
| GSE46505 |
Yeast X-Chromosome Associated Protein 5 (Xap5) Functions with H2A.Z to Suppress Aberrant Transcripts [ChIP-chip] |
| GSE46506 |
Yeast X-Chromosome Associated Protein 5 (Xap5) Functions with H2A.Z to Suppress Aberrant Transcripts |
|
