biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsJohn Thomsonmethylation profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-405385-mC is an epigenetic modification of the DNA base cytosine with roles in gene silencing events, imprinting and X inactivation. Phenobarbital (PB) is a well studied non-genotoxic carcinogen with roles in epigenetic perturbation. We profile 5mC in both control mouse livers as well as in the livers of 28 day PB treated mice. 5mC is largely found to reside in the bodies of active genes and promoter levels are perturbed upon PB induced gene activation events. 5-mC is assocated with promoters fo silent genes. Upon PB exposire a handfull of genes are induced which reveal decreases in promoter 5mC levels comparison of 5-mC profiles in 5 control and 5 PB exposed mouse liversbiostudies-arrayexpressGrowth Protocol - normal mouse liver tissueSample Processing - genomic DNA extracted from mouse liver following 28 day exposed to PhenobarbitalNucleic Acid Extraction - Genomic DNA (5mC-IP - 2.5 M-5g in 450 M-5l TE), sonicated to yield a fragment distribution of approximately 300-1000 bp, was denatured by incubation at 100M-0C for 10 min. Samples were rapidly chilled on wet-ice. At this point, 45 M-5l (10%) of denatured sample was removed and saved as input, and 45 M-5l 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 M-5g of M-oM-^AM-!-5mC (ActiveMotif; #39769) antibody were added to the remaining sample. Samples were incubated at 4C with gentle agitation overnight. Then, 40 M-5l of magnetic beads (DynabeadsM-. 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 M-5l 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 M-5l of digestion buffer (50 mM Tris pH 8.0, 10 mM EDTA , 0.5 % SDS) followed by addition of 10 M-5l of proteinase K (20 mg/ml; Roche Applied Science, Mannheim, Germany) and incubation at 52C for 1.5 hr with constant shaking (M-bM-^IM-%800 rpm). Finally, beads were removed using a magnetic rack and DNA was purified from the remaining sample using a QIAquickM-oM-^CM-^R PCR Purification Kit (QIAGEN,CA, USA), eluting in a final volume of 40 M-5l dH2O. Inputs were also purified using a QIAquickM-oM-^CM-^R PCR Purification Kit and eluted in 40 M-5l dH2O. Subsequently, 10 ng of input and IP DNA was subjected to whole genome amplification (WGA) using the GenomePlexM-. 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.Growth Protocol - PB exposed mouse liver tissueLabeling - Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)Hybridization - Labelled samples were applied commercially to a Nimblegen 2.1M Deluxe promoter array by Nimblegen, IcelandSample Processing - genomic DNA extracted from normal mouse liverMIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsJohn ThomsonJamie HackettFeature Extraction - Pair files normalised within arrays (loess), normalised between arrays (sclae normalisation), log2(IP/input)Image Adquisition - Arrays were scanned commercially by Nimblegen, IcelandAssay Data Transformation - ID_REF = VALUE = Pair files normalised within arrays (loess), normalised between arrays (sclae normalisation), log2(IP/input)falseIP of 5-methylcytosine (5-mC) enriched DNA fragments from control and PB treated mouse livers5-mC is an epigenetic modification of the DNA base cytosine with roles in gene silencing events, imprinting and X inactivation. Phenobarbital (PB) is a well studied non-genotoxic carcinogen with roles in epigenetic perturbation. We profile 5mC in both control mouse livers as well as in the livers of 28 day PB treated mice. 5mC is largely found to reside in the bodies of active genes and promoter levels are perturbed upon PB induced gene activation events. 5-mC is assocated with promoters fo silent genes. Upon PB exposire a handfull of genes are induced which reveal decreases in promoter 5mC levels comparison of 5-mC profiles in 5 control and 5 PB exposed mouse livers2012-09-12T00:00:00Z2023-09-14T10:25:47.955Z2022-03-15T15:05:41.385ZE-GEOD-40538GSE40538EFO_0002759