biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsJohn Thomsonmethylation profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-GEOD-413925-hmC 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 5hmC in both control mouse livers as well as in the livers of 7 day PB treated mice. 5hmC is largely found to reside in the bodies of active genes and promoter levels are perturbed upon PB induced gene activation events. 5-hmC is assocated with promoters fo silent genes. Upon PB exposire a handfull of genes are induced which reveal decreases in promoter 5hmC levels comparison of 5-hmC profiles in 5 control and 5 PB exposed mouse livers (dose: 7day PB)biostudies-arrayexpressGrowth Protocol - normal mouse liver tissueSample Processing - genomic DNA extracted from mouse liver following 7 day exposed to PhenobarbitalNucleic Acid Extraction - Genomic DNA (5hmC-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-!-5hmC (ActiveMotif; #39769) antibody were added to the remaining sample. Samples were incubated at 4M-oM-^BM-0C 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 4M-oM-^BM-0C 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 52M-oM-^BM-0C 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 DesignsHarri LempiM-CM-$inenJohn ThomsonFeature Extraction - Pair files normalised within arrays (loess), normalised between arrays (sclae normalisation), log2(IP/input)Assay Data Transformation - ID_REF = VALUE = Normalised data after normalised within arrays (loess) and between arrays (scale normalisation) resulting in log2(IP/input) scores for each probeImage Adquisition - Arrays were scanned commercially by Nimblegen, IcelandfalseIP of 5-methylcytosine (5-hmC) enriched DNA fragments from control and PB treated mouse livers (7 day)5-hmC 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 5hmC in both control mouse livers as well as in the livers of 7 day PB treated mice. 5hmC is largely found to reside in the bodies of active genes and promoter levels are perturbed upon PB induced gene activation events. 5-hmC is assocated with promoters fo silent genes. Upon PB exposire a handfull of genes are induced which reveal decreases in promoter 5hmC levels comparison of 5-hmC profiles in 5 control and 5 PB exposed mouse livers (dose: 7day PB)2012-11-10T00:00:00Z2023-08-29T13:27:54.104Z2021-10-08T11:17:32ZE-GEOD-41392GSE41392EFO_0002759