treatment: phenobarbital tissue: liver gender: M antibody: 5-hmC antibody manufacturer: Active Motif time: 28d
Treatment protocol
genomic DNA extracted from mouse liver following 28 day exposed to Phenobarbital
Growth protocol
PB exposed mouse liver tissue
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA (5hmC-IP - 2.5 µg in 450 µl TE), sonicated to yield a fragment distribution of approximately 300-1000 bp, was denatured by incubation at 100°C for 10 min. Samples were rapidly chilled on wet-ice. At this point, 45 µl (10%) of denatured sample was removed and saved as input, and 45 µl of 10X IP buffer (100 mM Na-Phosphate pH 7.0 (mono and dibasic), 1.4 M NaCl, 0.5 % Triton X-100) and 1 µg of -5hmC (ActiveMotif; #39769) antibody were added to the remaining sample. Samples were incubated at 4C with gentle agitation overnight. Then, 40 µl of magnetic beads (Dynabeads® Protein G, Invitrogen, UK) in 1X IP buffer were added to each sample to allow magnetic separation of the antibody from the unbound DNA using a magnetic tube rack. Samples were incubated at 4C for 1 hr with gentle agitation. Beads were collected with a magnetic rack and washed with 1000 µl of 1X IP buffer at RT for 10 min with gentle agitation; washing was repeated three times. Beads were collected with a magnetic rack and re-suspended in 250 µl of digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA , 0.5 % SDS) followed by addition of 10 µl of proteinase K (20 mg/ml; Roche Applied Science, Mannheim, Germany) and incubation at 52C for 1.5 hr with constant shaking (≥800 rpm). Finally, beads were removed using a magnetic rack and DNA was purified from the remaining sample using a QIAquick PCR Purification Kit (QIAGEN,CA, USA), eluting in a final volume of 40 µl dH2O. Inputs were also purified using a QIAquick PCR Purification Kit and eluted in 40 µl dH2O. Subsequently, 10 ng of input and IP DNA was subjected to whole genome amplification (WGA) using the GenomePlex® Complete Whole Genome Amplification Kit (Sigma-Aldrich, UK) as per the manufacturer's instructions. Amplified DNA was run on a 1.2% agarose gel to confirm consistency of fragment size between samples.
Label
Cy5
Label protocol
Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)
genomic DNA extracted from mouse liver following 28 day exposed to Phenobarbital
Growth protocol
PB exposed mouse liver tissue
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA (5hmC-IP - 2.5 µg in 450 µl TE), sonicated to yield a fragment distribution of approximately 300-1000 bp, was denatured by incubation at 100°C for 10 min. Samples were rapidly chilled on wet-ice. At this point, 45 µl (10%) of denatured sample was removed and saved as input, and 45 µl of 10X IP buffer (100 mM Na-Phosphate pH 7.0 (mono and dibasic), 1.4 M NaCl, 0.5 % Triton X-100) and 1 µg of -5hmC (ActiveMotif; #39769) antibody were added to the remaining sample. Samples were incubated at 4C with gentle agitation overnight. Then, 40 µl of magnetic beads (Dynabeads® Protein G, Invitrogen, UK) in 1X IP buffer were added to each sample to allow magnetic separation of the antibody from the unbound DNA using a magnetic tube rack. Samples were incubated at 4C for 1 hr with gentle agitation. Beads were collected with a magnetic rack and washed with 1000 µl of 1X IP buffer at RT for 10 min with gentle agitation; washing was repeated three times. Beads were collected with a magnetic rack and re-suspended in 250 µl of digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA , 0.5 % SDS) followed by addition of 10 µl of proteinase K (20 mg/ml; Roche Applied Science, Mannheim, Germany) and incubation at 52C for 1.5 hr with constant shaking (≥800 rpm). Finally, beads were removed using a magnetic rack and DNA was purified from the remaining sample using a QIAquick PCR Purification Kit (QIAGEN,CA, USA), eluting in a final volume of 40 µl dH2O. Inputs were also purified using a QIAquick PCR Purification Kit and eluted in 40 µl dH2O. Subsequently, 10 ng of input and IP DNA was subjected to whole genome amplification (WGA) using the GenomePlex® Complete Whole Genome Amplification Kit (Sigma-Aldrich, UK) as per the manufacturer's instructions. Amplified DNA was run on a 1.2% agarose gel to confirm consistency of fragment size between samples.
Label
Cy3
Label protocol
Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)
Hybridization protocol
Labelled samples were applied commercially to a Nimblegen 2.1M Deluxe promoter array by Nimblegen, Iceland
Scan protocol
Arrays were scanned commercially by Nimblegen, Iceland
Description
PB exposed mouse liver tissue
Data processing
Normalised data after normalised within arrays (loess) and between arrays (sclae normalisation) resulting in log2(IP/input) scores for each probe
