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.
strain: B6C3F1/Crl (C57BL/6 ♂ x C3H/He ♀) gender: male tissue: liver donor_id: 20 treatment: Phenobarbital
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
18786602 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)
Phenobarbital mediates an epigenetic switch at the constitutive androstane receptor (CAR) target gene Cyp2b10 in the liver of B6C3F1 mice [MeDIP array].
