|
| Status |
Public on Feb 08, 2011 |
| Title |
ZF4_H3K4me3_Repl1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3K4me3 ChIP DNA
|
| Organism |
Danio rerio |
| Characteristics |
cell line: ZF4
source: American Type Culture Collection
developmental stage: Embryo
chip antibody: anti-H3K4me3 from Diagenode (cat# pAb-003-050)
|
| Growth protocol |
Cell grown under standard condition as per Driever and Rangini, 1993 In vitro Cell Dev Biol.Anim 294, 749
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Histone ChIPs were done as described (Dahl, Collas, 2007) using 0.5 A260 units chromatin per ChIP. In short, cells were cross-linked in suspension with 1% formaldehyde for 8 min and quenched with glycine. Lysis buffer was added to ~120 µl and samples incubated for 5 min on ice. Cells were sonicated on ice using a Bioruptor (Diagenode) to produce fragments of ~400 bp. The lysate was centrifuged, the supernatant collected, chromatin diluted to 0.5 A260 units and 100 µl was incubated with 2.4 μg antibody coupled to Dynabeads Protein A (Invitrogen) for 2 h at 4oC. ChIP material was washed as described [46]. Elution buffer containing 1 % SDS and 50 µg/ ml Proteinase K was added and samples incubated for 2 h at 68oC on a Thermomixer (Eppendorf); after a second extraction both supernatants were pooled. ChIP DNA was purified by phenol-chloroform isoamylalcohol extraction, ethanol-precipitated and dissolved in 50 μl TE buffer. 5 µg (10 µl) RNase A (Roche) was added before ChIP DNA elution. ChIP DNA samples were dissolved in 16 μl MilliQ water. Input DNA (channel 2) was purified by phenol-chloroform isoamylalcohol extraction and RNAse-treated as above.
|
| Label |
Cy5
|
| Label protocol |
Labeling done by Nimblegen as per normal service protocol
|
| |
|
| Channel 2 |
| Source name |
Input DNA from ZF4 cells
|
| Organism |
Danio rerio |
| Characteristics |
cell line: ZF4
source: American Type Culture Collection
developmental stage: Embryo
|
| Growth protocol |
Cell grown under standard condition as per Driever and Rangini, 1993 In vitro Cell Dev Biol.Anim 294, 749
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Histone ChIPs were done as described (Dahl, Collas, 2007) using 0.5 A260 units chromatin per ChIP. In short, cells were cross-linked in suspension with 1% formaldehyde for 8 min and quenched with glycine. Lysis buffer was added to ~120 µl and samples incubated for 5 min on ice. Cells were sonicated on ice using a Bioruptor (Diagenode) to produce fragments of ~400 bp. The lysate was centrifuged, the supernatant collected, chromatin diluted to 0.5 A260 units and 100 µl was incubated with 2.4 μg antibody coupled to Dynabeads Protein A (Invitrogen) for 2 h at 4oC. ChIP material was washed as described [46]. Elution buffer containing 1 % SDS and 50 µg/ ml Proteinase K was added and samples incubated for 2 h at 68oC on a Thermomixer (Eppendorf); after a second extraction both supernatants were pooled. ChIP DNA was purified by phenol-chloroform isoamylalcohol extraction, ethanol-precipitated and dissolved in 50 μl TE buffer. 5 µg (10 µl) RNase A (Roche) was added before ChIP DNA elution. ChIP DNA samples were dissolved in 16 μl MilliQ water. Input DNA (channel 2) was purified by phenol-chloroform isoamylalcohol extraction and RNAse-treated as above.
|
| Label |
Cy3
|
| Label protocol |
Labeling done by Nimblegen as per normal service protocol
|
| |
|
| |
| Hybridization protocol |
Hybridization done by Nimblegen as per normal service protocol
|
| Scan protocol |
Scanning done by Nimblegen as per normal service protocol
|
| Data processing |
log2 (ChIP/Input) with biweight mean of values subtracted
|
| |
|
| Submission date |
Aug 18, 2010 |
| Last update date |
Feb 09, 2011 |
| Contact name |
Philippe Collas |
| Organization name |
University of Oslo
|
| Department |
Institute of Basic Medical Sciences
|
| Street address |
PO Box 1112 Blindern
|
| City |
Oslo |
| ZIP/Postal code |
0317 |
| Country |
Norway |
| |
|
| Platform ID |
GPL10835 |
| Series (2) |
| GSE23691 |
Mapping of H3K4 and H3K27 trimethylation in zebrafish cells (ChIP-chip) |
| GSE23872 |
Mapping of H3K4 and H3K27 trimethylation in zebrafish cells |
|
