|
| Status |
Public on Dec 16, 2011 |
| Title |
Liver_Control_5_rep1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Liver_Control_5_MeDIP
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6C3F1/Crl (C57BL/6 ♂ x C3H/He ♀)
gender: male
tissue: liver
donor_id: 5
treatment: Control
duration: 4 weeks
medip antibody: anti-5-Methylcytosine antibody
medip antibody vendor: Eurogentec
medip antibody cat#: MMS-900P-B
duration: 4 weeks
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA from liver and kidney tissue samples was prepared by overnight Proteinase K (pK) treatment in lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 100 mM NaCl, 0.5% SDS), phenol-chloroform extraction, ethanol precipitation and RNaseA digestion. Genomic DNA was sonicated (Bioruptor, Diagenode) to produce random fragments ranging in size from 300 to 1,000 bp and 6 µg of fragmented DNA was used for a MeDIP assay: DNA was denatured for 10 min at 95°C and immunoprecipitated for 2 hrs at 4°C with 10 µl of monoclonal antibody against 5-methylcytidine (Eurogentec) in a final volume of 500 µl IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100). The mixture was incubated with 60 µl magnetic beads (Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen) for 2 hrs at 4°C and washed three times with 700 µl of IP buffer. Beads were subsequently treated with pK for 3 hrs at 50°C and the methylated DNA recovered by phenol-chloroform extraction followed by ethanol precipitation. For microarray analysis the genomic input DNA and MeDIP enriched DNA was amplified using WGA2: GenomePlex Complete Whole Genome kit (Sigma) and 3 µg of DNA was sent to Roche Nimblegen (Madison, USA) for Cy3 and Cy5 labeling and hybridization on mouse promoter tiling arrays.
|
| Label |
cy5
|
| Label protocol |
Labeling of the immunoprecipitated (IP) and Input DNA samples with Cy5 and Cy3 respectively, using the NimbleGen Dual-Color DNA Labeling Kit.
|
| |
|
| Channel 2 |
| Source name |
Liver_Control_5_Input DNA
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6C3F1/Crl (C57BL/6 ♂ x C3H/He ♀)
gender: male
tissue: liver
donor_id: 5
treatment: Control
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA from liver and kidney tissue samples was prepared by overnight Proteinase K (pK) treatment in lysis buffer (10 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 100 mM NaCl, 0.5% SDS), phenol-chloroform extraction, ethanol precipitation and RNaseA digestion. Genomic DNA was sonicated (Bioruptor, Diagenode) to produce random fragments ranging in size from 300 to 1,000 bp and 6 µg of fragmented DNA was used for a MeDIP assay: DNA was denatured for 10 min at 95°C and immunoprecipitated for 2 hrs at 4°C with 10 µl of monoclonal antibody against 5-methylcytidine (Eurogentec) in a final volume of 500 µl IP buffer (10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100). The mixture was incubated with 60 µl magnetic beads (Dynabeads M-280 Sheep anti-mouse IgG (Invitrogen) for 2 hrs at 4°C and washed three times with 700 µl of IP buffer. Beads were subsequently treated with pK for 3 hrs at 50°C and the methylated DNA recovered by phenol-chloroform extraction followed by ethanol precipitation. For microarray analysis the genomic input DNA and MeDIP enriched DNA was amplified using WGA2: GenomePlex Complete Whole Genome kit (Sigma) and 3 µg of DNA was sent to Roche Nimblegen (Madison, USA) for Cy3 and Cy5 labeling and hybridization on mouse promoter tiling arrays.
|
| Label |
cy3
|
| Label protocol |
Labeling of the immunoprecipitated (IP) and Input DNA samples with Cy5 and Cy3 respectively, using the NimbleGen Dual-Color DNA Labeling Kit.
|
| |
|
| |
| Hybridization protocol |
Pooling and hybridization the samples to the arrays using a NimbleGen Hybridization Kit and NimbleGen Hybridization System 4 or 12. 5. Washing the arrays using the NimbleGen Wash Buffer Kit and the NimbleGen Microarray Dryer
|
| Scan protocol |
Scaning the array using the 2-μm, high-resolution NimbleGen MS 200 Microarray Scanner
|
| Description |
18412602
The two sources labeled cy5 and cy3 are from the same sample (two replicates for each sample)
|
| Data processing |
Image analysis with NimbleScan software, followed by M-value calculation (log2(cy5/cy3) of channels), loess smoothing M-values, averaging probes in promotors, median centering all scaling chips (see Lempiäinen et al., PLoS One. 2011 Mar 24), summarized per promotor (therefore probes of the same SEQ_ID entry have the same value per sample)
|
| |
|
| Submission date |
Dec 15, 2011 |
| Last update date |
Jun 08, 2015 |
| Contact name |
Jonathan Moggs |
| E-mail(s) |
jonathan.moggs@novartis.com
|
| Organization name |
Novartis
|
| Street address |
Fabrikstrasse 2
|
| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL7060 |
| Series (3) |
| GSE34462 |
Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice [MeDIP array]. |
| GSE34463 |
Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice. |
| GSE68387 |
IMI MARCAR Project: towards novel biomarkers for cancer risk assessment |
|
