|
| Status |
Public on Feb 09, 2013 |
| Title |
k27_ko_rep3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
input DNA for Dnmt1-/- MEFs for H3K27me3 rep3
|
| Organism |
Mus musculus |
| Characteristics |
background strain: unknown [Lande-Diner et al 2007 (PMID:17311920)]
cell type: Mouse embryonic fibroblasts
genotype: trp53-/- dnmt1-/-
chip antibody: None, input DNA
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
MEFs were cultured under standard conditions
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native ChIP was performed as described previously (Eskeland et al, 2010, Mol Cell, 38: 452-464) with the following adaptations. Protein-A coated Dynabeads (Invitrogen) were used throughout. Chromatin was digested with 25U of Mnase (micrococcal nuclease)(Worthington) for exactly 10min at room temperature. Following immunoprecipitation beads were washed three times for 10 min at 4°C in N-ChIP wash buffer (150mM NaCl; 10mM Tris pH8; 2mM EDTA; 1% NP40; 1% sodium deoxycholate w/v). Controls for N-ChIP experiments included a species matched IgG to control for background, negative and positive genomic regions to show specificity, and the use of input DNA to control for technical variations between cell lines or treatments. Antibodies were tested by western blot for recognition of histone-sized proteins only and by specific enrichment at positive control regions by N-ChIP. Enrichments were measured by qPCR.
|
| Label |
Cy3
|
| Label protocol |
Labelling was perfromed according to Nimblegen instructions by a Nimblegen service centre
|
| |
|
| Channel 2 |
| Source name |
ChIP DNA for Dnmt1-/- MEFs for H3K27me3 rep3
|
| Organism |
Mus musculus |
| Characteristics |
background strain: unknown [Lande-Diner et al 2007 (PMID:17311920)]
cell type: Mouse embryonic fibroblasts
genotype: trp53-/- dnmt1-/-
chip antibody: H3K27me3
antibody vendor: Upstate
antibody catalog number: 07-449
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
MEFs were cultured under standard conditions
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Native ChIP was performed as described previously (Eskeland et al, 2010, Mol Cell, 38: 452-464) with the following adaptations. Protein-A coated Dynabeads (Invitrogen) were used throughout. Chromatin was digested with 25U of Mnase (micrococcal nuclease)(Worthington) for exactly 10min at room temperature. Following immunoprecipitation beads were washed three times for 10 min at 4°C in N-ChIP wash buffer (150mM NaCl; 10mM Tris pH8; 2mM EDTA; 1% NP40; 1% sodium deoxycholate w/v). Controls for N-ChIP experiments included a species matched IgG to control for background, negative and positive genomic regions to show specificity, and the use of input DNA to control for technical variations between cell lines or treatments. Antibodies were tested by western blot for recognition of histone-sized proteins only and by specific enrichment at positive control regions by N-ChIP. Enrichments were measured by qPCR.
|
| Label |
Cy3
|
| Label protocol |
Labelling was perfromed according to Nimblegen instructions by a Nimblegen service centre
|
| |
|
| |
| Hybridization protocol |
Hybridisation was perfromed according to Nimblegen instructions by a Nimblegen service centre
|
| Scan protocol |
Scanning was perfromed according to Nimblegen instructions by a Nimblegen service centre
|
| Description |
comparison of Dnmt1+/+ vs Dnmt1-/- mouse embryonic fibroblasts
|
| Data processing |
Raw signals were normalised using the loess and scale methods of the limma R package. ChIP signals were normalised to input by calculating the log2 ratio of ChIP/Input signals.
|
| |
|
| Submission date |
Feb 07, 2013 |
| Last update date |
Feb 09, 2013 |
| Contact name |
James P Reddington |
| Organization name |
EMBL
|
| Street address |
Meyerhofstrasse 1
|
| City |
Heidelberg |
| ZIP/Postal code |
69115 Heidelberg |
| Country |
Germany |
| |
|
| Platform ID |
GPL16605 |
| Series (2) |
| GSE44149 |
Chip-chip for H3K27me3 and H3K4me3 in DNA methylation deficient mouse embryonic fibroblasts |
| GSE44278 |
Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of polycomb-target genes |
|
