|
| Status |
Public on Aug 22, 2008 |
| Title |
Chromosomal distribution of Cnp3 in cd60 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Chromatin-immunoprecipitated (ChIP) DNA for Cnp3
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
neocentric cell; h- leu1 ura4 cen1D::kanR cd60 Cnp3-GFP strain
|
| Biomaterial provider |
none
|
| Treatment protocol |
Exponentially growing fission yeast cells were asynchromously cultured at 33 °C in YES, and transferred to 18 °C for 2 h prior to fixation.
|
| Growth protocol |
Cells were fixed with 3% formaldehyde at 18 °C for 35 min, and the fixation reaction was terminated by adding 330 mM final concentration of glycine and incubating for 10 min; the cells were collected by centrifugation and washed 3 times with 1x PBS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell lysates were prepared by glass bead vortexing in FA-lysis buffer (50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1x Complete) containing 0.5% SDS. The cross-linked chromatin was then pelleted by centrifuging the lysate, and after a wash with ice-cold FA-lysis buffer containing 0.1% SDS, the chromatin pellet was sonicated to yield soluble fragments with an average size of 500 bp and a range between 300 and 1000 bp. The reaction was clarified by centrifugation, and the chromatin solution was supplemented with 1% Triton X-100 and applied for the immunoprecipitation using anti-GFP (Roche) and Dynabeads M-280 (Dynal). The beads were subsequently washed 3 times in ice-cold FA-lysis buffer containing 0.1% SDS and 1% Triton X-100; once in the same buffer containing 0.5 M NaCl; once in 10 mM Tris-HCl (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, and 0.5% sodium deoxycholate; and once in TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). To elute DNA and revert cross-links, the beads were incubated with SDS-TE (1% SDS, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA) for 15 h at 65 °C, followed by digestion with proteinase K at 42 °C. After extraction with phenol, DNA was precipitated with isopropanol and resuspended in TE.
|
| Label |
Cy5
|
| Label protocol |
ChIP-purified DNA was treated with RNase A, flush-ended using T7 DNA polymerase, and ligated with the double-stranded DNA linker using T4 DNA ligase. Linker-ligated DNA was PCR-amplified using AmpliTaq DNA polymerase, and then labelled with Cy5-dUTP by using the Agilent Genomic DNA Labeling Kit PLUS.
|
| |
|
| Channel 2 |
| Source name |
Chromatin-input DNA (WCE) for Cnp3
|
| Organism |
Schizosaccharomyces pombe |
| Characteristics |
neocentric cell; h- leu1 ura4 cen1D::kanR cd60 Cnp3-GFP strain
|
| Biomaterial provider |
none
|
| Treatment protocol |
Exponentially growing fission yeast cells were asynchromously cultured at 33 °C in YES, and transferred to 18 °C for 2 h prior to fixation.
|
| Growth protocol |
Cells were fixed with 3% formaldehyde at 18 °C for 35 min, and the fixation reaction was terminated by adding 330 mM final concentration of glycine and incubating for 10 min; the cells were collected by centrifugation and washed 3 times with 1x PBS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell lysates were prepared by glass bead vortexing in FA-lysis buffer (50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1x Complete) containing 0.5% SDS. The cross-linked chromatin was then pelleted by centrifuging the lysate, and after a wash with ice-cold FA-lysis buffer containing 0.1% SDS, the chromatin pellet was sonicated to yield soluble fragments with an average size of 500 bp and a range between 300 and 1000 bp. The reaction was clarified by centrifugation, and the chromatin solution was supplemented with 1% Triton X-100. To elute DNA and revert cross-links, the beads were incubated with SDS-TE (1% SDS, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA) for 15 h at 65 °C, followed by digestion with proteinase K at 42 °C. After extraction with phenol, DNA was precipitated with isopropanol and resuspended in TE.
|
| Label |
Cy3
|
| Label protocol |
WCE DNA was treated with RNase A, flush-ended using T7 DNA polymerase, and ligated with the double-stranded DNA linker using T4 DNA ligase. Linker-ligated DNA was PCR-amplified using AmpliTaq DNA polymerase, and then labelled with Cy3-dUTP by using the Agilent Genomic DNA Labeling Kit PLUS.
|
| |
|
| |
| Hybridization protocol |
Hybridisation was carried out at 65 °C for 40 h against the 4 x 44K slide of S. pombe whole genome ChIP-on-chip microarray (G4810A, Agilent Technologies) according to the manufacturer’s instructions. The slide was then washed with Wash Buffer 1 for 5 min at room temperature and with Wash Buffer 2 for 5 min at 31 ºC, according to the manufacturer’s instructions (Agilent Technologies), in ozone-depleted surroundings.
|
| Scan protocol |
Microarray slide was scanned using the Agilent Microarray Scanner G2505B and Scan Control Software G2565BA, followed by extraction of information from the probe features using Agilent Feature Extraction Software 9.5.1.1.
|
| Description |
Comparing between genome DNA (Cy3) and DNA co-immunopurified with Cnp3 (Cy5) from the Schizosaccharomyces pombe cen1 deletion survivor cd60
|
| Data processing |
For each data set, intra-array Lowess (intensity-dependent) normalization was performed using the Agilent ChIP Analytics Software 1.3.1, and the enrichment ratios were calculated by dividing the normalised ChIP (Cy5) signals over corresponding WCE (Cy3) signals.
|
| |
|
| Submission date |
Jan 26, 2008 |
| Last update date |
Oct 30, 2013 |
| Contact name |
Kojiro Ishii |
| Organization name |
Kochi University of Technology
|
| Street address |
185 Miyanokuchi, Tosayamada
|
| City |
Kami |
| State/province |
Kochi |
| ZIP/Postal code |
782-8502 |
| Country |
Japan |
| |
|
| Platform ID |
GPL16218 |
| Series (1) |
| GSE10287 |
Heterochromatin integrity affects chromosome reorganization after centromere dysfunction |
|
