|
| Status |
Public on Dec 02, 2011 |
| Title |
postMBT_H3K4me3 Repl2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
postMBT H3K4me3 ChIP DNA
|
| Organism |
Danio rerio |
| Characteristics |
cell type: Embryonic cells
source: AB strain zebrafish embryos
developmental stage: postMBT, 5.3 hpf, 50% epiboly
chip antibody: H3K4me3
vendor: Diagenode pAb-003-050
|
| Growth protocol |
Fish and embryos grown as per AAALAC certification
|
| 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, dechorionated embryos were cross-linked with 1% formaldehyde for 8 min and quenched with glycine. Lysis buffer was added and samples incubated for 5 min on ice. Cells were sonicated on ice using a Bioruptor (Diagenode) to produce fragments of 400 bp or less. The lysate was centrifuged, the supernatant collected, and chromatin was incubated with 2.4 μg antibody coupled to Dynabeads Protein A (Invitrogen) for 2 h ro overnight at 4oC. ChIP material was washed, 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 |
postMBT H3K4me3 Input DNA
|
| Organism |
Danio rerio |
| Characteristics |
cell type: Embryonic cells
source: AB strain zebrafish embryos
developmental stage: postMBT, 5.3 hpf, 50% epiboly
antibody: none
|
| Growth protocol |
Fish and embryos grown as per AAALAC certification
|
| 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, dechorionated embryos were cross-linked with 1% formaldehyde for 8 min and quenched with glycine. Lysis buffer was added and samples incubated for 5 min on ice. Cells were sonicated on ice using a Bioruptor (Diagenode) to produce fragments of 400 bp or less. The lysate was centrifuged, the supernatant collected, and chromatin was incubated with 2.4 μg antibody coupled to Dynabeads Protein A (Invitrogen) for 2 h ro overnight at 4oC. ChIP material was washed, 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 |
Feb 14, 2011 |
| Last update date |
Dec 02, 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 (1) |
| GSE27314 |
Marking of the genome by H3K4 methylation prior to embryonic gene activation |
|
