Comment[ArrayExpressAccession]	E-GEOD-41392
MAGE-TAB Version	1.1									
Public Release Date	2012-11-10
Investigation Title	IP of 5-methylcytosine (5-hmC) enriched DNA fragments from control and PB treated mouse livers (7 day)									
Comment[Submitted Name]	IP of 5-methylcytosine (5-hmC) enriched DNA fragments from control and PB treated mouse livers (7 day)
Experiment Description	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)									
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl								
Person Last Name	Thomson	LempiM-CM-$inen	Thomson							
Person First Name	John	Harri	John							
Person Mid Initials	Paterson		P							
Person Email	john.thomson@hgu.mrc.ac.uk									
Person Affiliation	Medical Research Council									
Person Address	Human genetics Unit, Medical Research Council, Crewe Road, Edinburgh, United Kingdom									
Person Roles	submitter									
Protocol Name	P-GSE41392-1	P-GSE41392-6	P-GSE41392-3	P-GSE41392-10	P-GSE41392-8	P-GSE41392-7	P-GSE41392-9	P-GSE41392-2	P-GSE41392-4	P-GSE41392-5
Protocol Description	ID_REF =  VALUE = Normalised data after normalised within arrays (loess) and between arrays (scale normalisation) resulting in log2(IP/input) scores for each probe	Labelled samples were applied commercially to a Nimblegen 2.1M Deluxe promoter array by Nimblegen, Iceland	normal mouse liver tissue	PB exposed mouse liver tissue	Pair files normalised within arrays (loess), normalised between arrays (sclae normalisation), log2(IP/input)	Arrays were scanned commercially by Nimblegen, Iceland	genomic DNA extracted from mouse liver following 7 day exposed to Phenobarbital	genomic DNA extracted from normal mouse liver	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.	Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)
Protocol Type	bioassay_data_transformation	hybridization	grow	grow	feature_extraction	image_aquisition	specified_biomaterial_action	specified_biomaterial_action	nucleic_acid_extraction	labeling
Experimental Factor Name	TREATMENT	ANTIBODY								
Experimental Factor Type	treatment	antibody								
Comment[SecondaryAccession]	GSE41392									
Comment[GEOReleaseDate]	2012-11-10									
Comment[ArrayExpressSubmissionDate]	2012-10-05									
Comment[GEOLastUpdateDate]	2012-11-11									
Comment[AEExperimentType]	methylation profiling by array									
Comment[AdditionalFile:Data1]	GSE41392_hmedip_liver_7day_PB.txt									
SDRF File	E-GEOD-41392.sdrf.txt