{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Colm Nestor"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-3176"],"description":["Comparison of the methylation profile of mouse embryonic fibroblasts (MEFs) before and during adaptation to cell culture conditions. Matched expression data is deposited under accession:  E-MTAB-3172"],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - Genomic DNA was extracted from cells using TRIzol. Genomic DNA (2.5 µg in 450 µL of TE), sonicated to yield a fragment distribution of 300–1000 bp, was denatured by incubation for 10 min at 100°C.  Samples were rapidly chilled on wet ice. At this  point, 45 µL (10%)  of denatured sample was re- moved and saved as input, and 45 µL of 10X IP buffer (100 mM Na- Phosphate at pH 7.0 [mono and  dibasic], 1.4 M NaCl, 0.5% Triton X-100)  and  1 µg of α-5hmC (diagenode) antibody were added to the remaining sample. Samples were incubated overnight at 4°C with gentle agitation. Then, 40 µL of magnetic beads (Dynabeads Protein G; Invitrogen) in 1X IP buffer was added to each sample to allow magnetic separation of the antibody from the unbound DNA using a magnetic tube rack.  Samples were incubated for 1 h at 4°C with gentle agitation. Beads were collected with a magnetic rack and washed with 1 mL of 1X IP buffer for 10 min at room temperature with gentle agitation; washing was repeated three times. Beads  were  collected with a magnetic rack and resuspended in 250 µL of digestion buffer  (50 mM  Tris at pH 8.0,  10  mM  EDTA, 0.5%  SDS) followed by addition of 10 µL of proteinase K (20 mg/mL; Roche  Applied Science)  and  incubation overnight at 52°C 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), eluting in a final volume of 47 µL of dH2O. Inputs were also purified using a QIAquick PCR Purification Kit and eluted in 47 µL of 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) as per the manufacturer’s instructions.","Scaning - Arrays were scanned on a NimbleGen MS200 scanner per manufacturer's protocol (http://www.nimblegen.com/).","Growth Protocol - Mouse embryonic fibroblast (MEF) cultures were established from wild-type, B6 13.5 dpc embryos. The excised uterus containing the embryos was transferred to a 25 mL universal tube containing cold PBS/PS solution (1X phosphate buffered saline and 5% penicillin/streptomycin). The embryo string was transferred to a 10 cm petri dish containing 5ml PBS/PS solution and the placenta, membranes, string and organs were discarded. The resulting material was finely chopped with a scalpel blade and incubated in 300 µl trypsin per embryo for 10 min at 37ºC. The tissue was further homogenised by repeated (20 times) syringing (19G needle and 3 mL syringe). After the addition of 5 mL growth medium (Dulbecco's Modified Eagle Medium Invitrogen, cat. # 41965-039; 15% Feotal calf serum; 5% penicillin/streptomycin; 5% sodium pyruvate solution, Invitogen cat.#: S8636; 5% MEM Non-essential Amino Acid Solution, Invitrogen cat. #: M7145), cells were briefly centrifuged (300 x g for 4 mins) and re-suspended in 5 mL growth media and plated onto T25 cell culture flask.","Hybridization - Standard hyb conditions as per Nimblegen's protocol","Labeling - Amplified DNA samples were Cy5- (IP) or Cy3- (Input) labelled by random priming using the Dual-Color DNA Labeling Kit (NimbleGen)."],"figure_sub":["MIAME Score","Raw Data","Organization","Assays and Data","MAGE-TAB Files","Array Designs"],"data_protocol":["Data Transformation - Images were processed using Nimblegen's standard protocol for Nimblescan DNA methylation DNA extraction. Normalised within arrays (loess), final value log2(IP/input)"],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"study_type":["methylation profiling by array"],"species":["Mus musculus"],"pubmed_title":["Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems"],"pubmed_authors":["Richard Meehan","Colm E. Nestor, Raffaele Ottaviano, Diana Reinhardt, Hazel Cruickshanks , Heidi Mjoseng, Rhoanne C. McPherson, Antonio Lentini, John P. Thomson, Donncha S. Dunican, Sari Pennings, Stephen M. Anderton, Mikael Benson, Richard R. Meehan","Colm Nestor"],"additional_accession":[]},"is_claimable":false,"name":"Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems","description":"Comparison of the methylation profile of mouse embryonic fibroblasts (MEFs) before and during adaptation to cell culture conditions. Matched expression data is deposited under accession:  E-MTAB-3172","dates":{"release":"2014-12-17T00:00:00Z","modification":"2022-02-03T05:33:11.408Z","creation":"2022-02-03T05:33:11.408Z"},"accession":"E-MTAB-3176","cross_references":{"EFO":["EFO_0002944","EFO_0003814","EFO_0003813","EFO_0003789","EFO_0002759","EFO_0003816","EFO_0003815"]}}