|
| Status |
Public on Apr 24, 2007 |
| Title |
ChIP-Chip with anti-Flag antibody, replicate 1 of 2. |
| Sample type |
genomic |
| |
|
| Source name |
Cultured Drosophila S2 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
Cultured Drosophila S2 cells
|
| Treatment protocol |
S2 cells were transfected with pMT-Flag-cwo (1.9 ug) or pMT-Flag (1.9 ug) and pCoHygro (0.1 ug, Invitrogen, for selection) using Effectene (QIAGEN) according to the manufacturer’s instructions. Hygromycin-B (300 ug /ml final concentration, Invitrogen) was added 5 days after transfection. After 3 weeks, the established stable cell lines were used for the ChIP assay. 4×10^7 cells were collected and stimulated by copper sulfate to induce fusion genes.
|
| Growth protocol |
Drosophila S2 cells were maintained in Schneider′s Drosophila medium (Invitrogen) supplemented with 10% FBS (Bioserum) and antibiotics (12.5 U/ml penicillin, 12.5 mg/ml streptomycin; GIBCO).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were harvested 24 h after stimulation. ChIP was then performed according to the previously described method(Yamashita, M. et al. 2002. Identification of a conserved GATA3 response element upstream proximal from the interleukin-13 gene locus. J Biol Chem 277(44): 42399-42408) with some modifications. In this report, we employed Chromatin Immunoprecipitation Assay Kits (Upstate Biotechnology), anti-Flag M2 antibody (SIGMA) and anti-V5 antibody (SIGMA). Protein G-Sepharose (Amersham) was used for precipitation of antibody/protein immune complexes. The resulting precipitated DNA was amplified by adaptor-mediated PCR(Cawley et al. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116(4): 499-509) and then fragmented to 50-150 bp by DNase I (Epicentre) in One-Phor-All buffer (Pharmacia). The distribution of fragmented DNA size was verified on a 2% agarose gel.
|
| Label |
Biotin
|
| Label protocol |
The fragmented DNA was then end-labeled with GeneChip Labeling Reagent (Affymetrix) by using terminal deoxynucleotidyltransferase (Roche) in TdT buffer (Roche) and CoCl2 (Roche). To prepare the hybridization cocktail, 5 ug of labeled DNA fragment was mixed with final 1x MES, 3 M TMAC, 0.02% Triton (SIGMA), 50 pM Oligo B2, 1×Hybridization Control (Affymetrix), and 100 ug/ul Herring Sperm DNA (Promega).
|
| |
|
| Hybridization protocol |
200 ul Hybridization cocktail was applied to Drosophila genome tiling array (Affymetrix) and hybridized at 45 dC for 18 h with 45 rpm rotation. Stain and wash was executed by Fluidics station 450 with EukGE-WS2 ver.4 program.
|
| Scan protocol |
Arrays were scanned by GeneChip Scanner 7G (Affymetrix).
|
| Description |
Data from the chromatin immunoprecipitation with anti-Flag antibody on Drosophila genome tiling array. Replicate no.1 of 2 (Independent sampling).
|
| Data processing |
Raw data (CEL file) was normalized and analyzed by TileMap (Ji, H. and Wong, W.H. 2005. TileMap: create chromosomal map of tiling array hybridizations. Bioinformatics 21(18): 3629-3636.) with its default parameters (See suplemental file: TileMap_parameterfiles.zip).
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| |
|
| Submission date |
Apr 22, 2007 |
| Last update date |
Apr 23, 2007 |
| Contact name |
Rikuhiro G Yamada |
| E-mail(s) |
rikuhiro@cdb.riken.jp
|
| Phone |
+81-78-306-3191
|
| Fax |
+81-78-306-3194
|
| Organization name |
RIKEN
|
| Department |
Center for Developmental Biology
|
| Lab |
Laboratory for Systems Biology
|
| Street address |
Minatojima-Minamimachi 2-2-3
|
| City |
Kobe |
| State/province |
Hyogo |
| ZIP/Postal code |
650-0047 |
| Country |
Japan |
| |
|
| Platform ID |
GPL3923 |
| Series (1) |
| GSE7569 |
Chromatin immunoprecipitation (ChIP) assay of CWO protein with Drosophila genome tiling array |
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