|
| Status |
Public on Jun 01, 2009 |
| Title |
H3K9ac_against_TotalH3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3K9ac ChIP DNA from EL4 cells
|
| Organism |
Mus musculus |
| Characteristics |
Antibody: H3K9ac
Cell Line: EL4
Treatment: Unstimulated
Time: 0h
|
| Treatment protocol |
EL4 cells were stimulated with PMA (10 ng/ml phorbol myristate acetate) and Ionomcyin (1 microM) for 0h, 0.5h or 4h
|
| Growth protocol |
EL-4 were cultured in RPMI 1640 medium with 10 mM HEPES, 10% fetal calf serum (FCS; CSL, Australia), 120 microg/ml penicillin, and 16 microg/ml gentamycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were performed as previously described (Chen et al. Mol. Cell. Biol (2005) 25:3209) with some modifications. Briefly, cells were harvested and cross-linked with formaldehyde. Cells were lysed and then sonicated using the Bioruptor (Diagenode) to give fragments between 200bp to 1000bp lengths. Samples were precleared with protein-A agarose/salmon sperm DNA beads (Upstate) then immunoprecipitated with 2.5 microg anti-histone-H3 (Abcam), 4 microg anti-acetyl-H3K9 (Upstate) or mock immunoprecipiatated. Immune complexes were recovered using salmon sperm DNA/protein A-agarose, washed and eluted. Following cross-link reversal and proteinase K treatment immunoprecipitated DNA was purified by phenol–chloroform extraction and ethanol precipitation.10ng ChIP DNA was amplified with the Whole Genome Amplification (WGA) kit from Sigma as per manufacturers instructions but with incorporation of dUTP.
|
| Label |
biotin
|
| Label protocol |
Samples (7.5μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| Channel 2 |
| Source name |
Pan Histone H3 ChIP DNA from EL4 cells
|
| Organism |
Mus musculus |
| Characteristics |
Antibody: Histone H3
Cell Line: EL4
Treatment: Unstimulated
|
| Treatment protocol |
EL4 cells were stimulated with PMA (10 ng/ml phorbol myristate acetate) and Ionomcyin (1 microM) for 0h, 0.5h or 4h
|
| Growth protocol |
EL-4 were cultured in RPMI 1640 medium with 10 mM HEPES, 10% fetal calf serum (FCS; CSL, Australia), 120 microg/ml penicillin, and 16 microg/ml gentamycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were performed as previously described (Chen et al. Mol. Cell. Biol (2005) 25:3209) with some modifications. Briefly, cells were harvested and cross-linked with formaldehyde. Cells were lysed and then sonicated using the Bioruptor (Diagenode) to give fragments between 200bp to 1000bp lengths. Samples were precleared with protein-A agarose/salmon sperm DNA beads (Upstate) then immunoprecipitated with 2.5 microg anti-histone-H3 (Abcam), 4 microg anti-acetyl-H3K9 (Upstate) or mock immunoprecipiatated. Immune complexes were recovered using salmon sperm DNA/protein A-agarose, washed and eluted. Following cross-link reversal and proteinase K treatment immunoprecipitated DNA was purified by phenol–chloroform extraction and ethanol precipitation.10ng ChIP DNA was amplified with the Whole Genome Amplification (WGA) kit from Sigma as per manufacturers instructions but with incorporation of dUTP.
|
| Label |
biotin
|
| Label protocol |
Samples (7.5μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| |
| Hybridization protocol |
Approximately 7.5μg of DNA was hybridzed per array using the Affymetrix hybridization kit. Arrays were hybridized for 16 hours at 45° at 60rpm using an Affymetrix hybridization oven and washed with Fluidics station 450 protocol FS450_0001
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G
|
| Description |
H3K4ac ChIP in Non-stimulated EL4 cells, using Total Histone 3 ChIP as control, 3 biological replicates
|
| Data processing |
All analysis was performed with NCBI build 36 of the mouse genome. The model-based analysis of tiling array (MAT) algorithm (Johnson WE et al PNAS 2006) was used to find regions of H3K9Ac with a bandwidth of 250bp and a max gap of 150bp. Either the matching total input DNA or H3 chips (as stated) were used as control samples.
|
| |
|
| Submission date |
Oct 20, 2008 |
| Last update date |
Jan 04, 2012 |
| Contact name |
Kristine Hardy |
| E-mail(s) |
kristine.hardy@anu.edu.au
|
| Organization name |
University of Canberra
|
| Lab |
Cytokine Gene Expression
|
| Street address |
University of Canberra
|
| City |
Bruce |
| State/province |
ACT |
| ZIP/Postal code |
0200 |
| Country |
Australia |
| |
|
| Platform ID |
GPL5811 |
| Series (2) |
| GSE13277 |
ChIP-on-chip examination of inducible genes in T cells |
| GSE13279 |
Defining the chromatin signature of inducible genes in T cells |
|
