|
| Status |
Public on Mar 09, 2010 |
| Title |
Otx ChIP-chip on Ciona intestinalis embryo (early gastrula) Sample 1, Array 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Whole Cell Extract (WCE) DNA
|
| Organism |
Ciona intestinalis |
| Characteristics |
fraction: input
antibody: none
|
| Treatment protocol |
Approximately 3.0 kb of the Otx promoter and the coding region of Otx were cloned into pDONR P4-P1R and pDONR221 (Invitrogen), respectively. cDNA encoding GFP was cloned into pDONR P2R-P3. Recombination of these three plasmids with Multisite Gateway technology (Invitrogen) resulted in a construct encoding GFP-tagged Otx. Fertilized eggs were electroporated with this DNA construct.
|
| Growth protocol |
C. intestinalis adults were obtained from the national bio-resource project for Ciona intestinalis and were maintained under constant light to induce oocyte maturation. Eggs and sperm were obtained surgically from the gonoduct. Following insemination, eggs were washed and maintained in seawater at 18˚C in Millipore-filtered seawater containing 50ug/ml streptomycin sulfate.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The embryo were fixed with 1% formaldehyde for 15 min. Chromatin was sonicated to obtain DNA fragments with an average of size of 1kb. After reversal of the fixation, the WCE DNA were amplified by LM-PCR.
|
| Label |
Cy3
|
| Label protocol |
Amplified DNA was directly labeled by random-primed, Klenow-based extension protocol that is used with CGH Labeling kit (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
GFP-tagged Otx ChIP in Ciona intestinalis embryo (early gastrula)
|
| Organism |
Ciona intestinalis |
| Characteristics |
fraction: IP
embryo: earyl gastrula
construct: GFP-tagged Otx
|
| Treatment protocol |
Approximately 3.0 kb of the Otx promoter and the coding region of Otx were cloned into pDONR P4-P1R and pDONR221 (Invitrogen), respectively. cDNA encoding GFP was cloned into pDONR P2R-P3. Recombination of these three plasmids with Multisite Gateway technology (Invitrogen) resulted in a construct encoding GFP-tagged Otx. Fertilized eggs were electroporated with this DNA construct.
|
| Growth protocol |
C. intestinalis adults were obtained from the national bio-resource project for Ciona intestinalis and were maintained under constant light to induce oocyte maturation. Eggs and sperm were obtained surgically from the gonoduct. Following insemination, eggs were washed and maintained in seawater at 18˚C in Millipore-filtered seawater containing 50ug/ml streptomycin sulfate.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The embryo were fixed with 1% formaldehyde for 15 min. Chromatin was sonicated to obtain DNA fragments with an average of size of 1kb. The DNA fragments were enriched by immunoprecipitation with an polyclonal antibody specific for GFP (Invitrogen). After reversal of the fixation, the immunoprecipitated DNA were amplified by LM-PCR.
|
| Label |
Cy5
|
| Label protocol |
Amplified DNA was directly labeled by random-primed, Klenow-based extension protocol that is used with CGH Labeling kit (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
Hybridization and washing protocols were according to the manufacturer’s instructions (Agilent Technologies).
|
| Scan protocol |
The microarrays were scanned with an Agilent G2565BA Microarray Scanner (Agilent Technologies).
|
| Description |
Otx ChIP-chip on Ciona intestinalis embryo (early gastrula) Sample 1, Array 2 GFP-tagged Otx ChIP-chip in Whole Genome region in Ciona intestinais embryo (early gastrula) using polyclonal anti-GFP antibody (Invitrogen)
|
| Data processing |
The resulting fluorescence intensity for each spot was quantified using Agilent Feature Extraction Software ver. 9.1 (Agilent Technologies).
|
| |
|
| Submission date |
Aug 17, 2009 |
| Last update date |
Mar 09, 2010 |
| Contact name |
Atsushi Kubo |
| Organization name |
Kyoto University
|
| Department |
Department of zoology, Graduate School of Science
|
| Street address |
Sakyo-ku
|
| City |
Kyoto |
| State/province |
Kyoto |
| ZIP/Postal code |
606-8502 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6556 |
| Series (2) |
| GSE17690 |
Otx ChIP-chip on Ciona intestinalis embryo (early gastrula) |
| GSE17976 |
ChIP-chip analysis of the Ciona intestinalis embryo |
|
