{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["John Thomson"],"study_type":["methylation profiling by array"],"organism":["Mus musculus"],"species":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-GEOD-40540"],"description":["This SuperSeries is composed of the following subset Series: GSE40537: IP of 5-hydroxymethylcytosine (5-hmC) enriched DNA fragments from control and PB treated mouse livers GSE40538: IP of 5-methylcytosine (5-mC) enriched DNA fragments from control and PB treated mouse livers GSE40773: Dynamic changes in liver 5M-bM-^@M-^P hydroxymethylcytosine profiles upon nonM-bM-^@M-^P genotoxic carcinogen exposure Refer to individual Series"],"repository":["biostudies-arrayexpress"],"sample_protocol":["Hybridization - The arrays were washed and stained with the GeneChipM-. Hybridization Wash and Stain kit (Affymetrix). The washing and staining steps were performed with GeneChipM-. Fluidics Workstation 450 (Affymetrix).","Sample Processing - Phenobarbital (Sigma 04710, 0.05% (w/v) in drinking water) was administered to one group through ad libitum access to drinking water for 28 days","Sample Processing - genomic DNA extracted from mouse liver following 28 day exposed to Phenobarbital","Nucleic 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.","Labeling - Processing of GeneChipM-. experiments was conducted as recommended by the manufacturer of the GeneChipM-. system (Affymetrix). For tissue samples, double stranded cDNA was synthesized with a starting amount of 0.1 M-5g total RNA. For RNA reverse transcription, the GeneChipM-. 3M-bM-^@M-2 IVT Express Labelling Assay (lot ID 0904012, Affymetrix) was used in the presence of a T7-(dT)24 DNA oligonucleotide primer (Affymetrix). The cDNA was then transcribed in vitro in the presence of biotinylated ribonucleotides to form biotin-labelled amplified RNA (aRNA). The labelled aRNA was then purified and quantified by UV spectrophotometry at 260 nm and fragmented","Growth Protocol - PB exposed mouse liver tissue","Labeling - Amplification of DNA samples was carried out commercially by NimbleGen, Iceland (Cy5- (IP) or Cy3- (Input) labelled)","Growth Protocol - normal mouse liver tissue","Nucleic Acid Extraction - Frozen liver and kidney samples were homogenized in TRIzol reagent (Invitrogen) and subsequently purified on a silica-gel-based-membrane (RNeasy, Qiagen) according to the manufacturer's instructions. RNA quality was assessed by measuring the RIN (RNA Integrity Number) using an Agilent 2100 Bioanalyzer. RNA was stored at M-bM-^HM-^R80M-0C until GeneChipM-. experiment analysis.","Nucleic 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 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 - 29M-bM-^@M-^S32 days old male B6C3F1/Crl (C57BL/6 M-bM-^YM-^B x C3H/He M-bM-^YM-^@) mice were obtained from Charles River Laboratories (Germany). Animals were allowed to acclimatise for 5 days prior to being randomly divided into two treatment groups of five animals each. Phenobarbital (Sigma 04710, 0.05% (w/v) in drinking water) was administered to one group through ad libitum access to drinking water for 28 days. Mice were checked daily for activity and behaviour and sacrificed on the last day of dosing (day 28)","Hybridization - Labelled samples were applied commercially to a Nimblegen 2.1M Deluxe promoter array by Nimblegen, Iceland","Sample Processing - genomic DNA extracted from normal mouse liver"],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","Processed Data","MAGE-TAB Files","Array Designs"],"pubmed_authors":["Richard Meehan","John Thomson","Jonathan Moggs","Arne MuM-LM-^Hller","Jamie Hackett","Harri LempiaM-LM-^Hinen"],"data_protocol":["Feature Extraction - Pair files normalised within arrays (loess), normalised between arrays (sclae normalisation), log2(IP/input)","Image Adquisition - Arrays were scanned commercially by Nimblegen, Iceland","Feature Extraction - The Affymetrix GeneChipM-. Operating Software (GCOS) was used to generate the primary and secondary raw data files.","Assay Data Transformation - ID_REF =  VALUE = Normalised data after normalised within arrays (loess) and between arrays (sclae normalisation) resulting in log2(IP/input) scores for each probe","Assay Data Transformation - ID_REF =  VALUE = RMA normalized and summarized values (log2 based)","Feature Extraction - Normalised data after normalised within arrays (loess) and between arrays (sclae normalisation) resulting in log2(IP/input) scores for each probe","Image Adquisition - Arrays were scanned using a solid-state laser scanner (GeneArrayM-. Scanner 3000 combined with the GeneChipM-. autoloader, Affymetrix).","Assay Data Transformation - ID_REF =  VALUE = Pair files normalised within arrays (loess), normalised between arrays (sclae normalisation), log2(IP/input)"],"additional_accession":[]},"is_claimable":false,"name":"IP of 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) enriched DNA fragments from control and PB treated mouse livers","description":"This SuperSeries is composed of the following subset Series: GSE40537: IP of 5-hydroxymethylcytosine (5-hmC) enriched DNA fragments from control and PB treated mouse livers GSE40538: IP of 5-methylcytosine (5-mC) enriched DNA fragments from control and PB treated mouse livers GSE40773: Dynamic changes in liver 5M-bM-^@M-^P hydroxymethylcytosine profiles upon nonM-bM-^@M-^P genotoxic carcinogen exposure Refer to individual Series","dates":{"release":"2012-09-12T00:00:00Z","modification":"2023-09-01T05:09:03.503Z","creation":"2022-03-07T01:17:23.015Z"},"accession":"E-GEOD-40540","cross_references":{"GEO":["GSE40540"],"EFO":["EFO_0002759"]}}