Comment[ArrayExpressAccession] E-GEOD-38505 Public Release Date 2012-06-05 Investigation Title Promoter methylation analysis in rat Pubertal Hypothalamus Comment[Submitted Name] Promoter methylation analysis in rat Pubertal Hypothalamus Experiment Description Promoter methylation analysis of hypothalamc DNA from female rats at different juvenile developmental reproductive stages. Results provide insight into the role of the hypothalamus in controlling the onset of puberty. SD rats were housed (4/cage) in a controlled environment and euthanized at different ages (Early Juvenile: 21 days, Late Juvenile: 28 days, Late Proestus (the day of first ovulation): 31 days. Rats were anesthetized and brains were rapidly removed. The hypothalamus was dissected away from the rest of the brain and flash frozen. Total RNA and DNA was isolated from each sample using AllPrep DNA/RNA Mini Kit-Qiagen (Valencia, CA). Date of Experiment Term Source Name EFO Term Source Version Term Source File http://www.ebi.ac.uk/efo/efo.owl Person Last Name Lomniczi Lomniczi Ojeda Person First Name Alejandro Alejandro Sergio Person Mid Initials R Person Email lomniczi@ohsu.edu Person Affiliation Oregon National Primate Research Center - OHSU Person Phone 503-690-5305 Person Fax 503-690-5384 Person Address Neuroscience, Oregon National Primate Research Center - OHSU, 505 NW 185th Ave, Beaverton, OR, USA Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE38505-1 P-GSE38505-5 P-GSE38505-4 P-GSE38505-2 P-GSE38505-3 P-GSE38505-6 Protocol Description ID_REF =
VALUE = scaled, log2 (ChIP/Input) ratio Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html). The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html) Rats were anesthetized and brains were rapidly removed. The hypothalamus was dissected away from the rest of the brain and flash frozen. Total RNA and DNA was isolated from each sample using AllPrep DNA/RNA Mini Kit-Qiagen (Valencia, CA). 2000ng of genomic DNA was digested over night with MseI. After Qiagen PCR cleanup, 150 ng of DNA were run in a 1% agarose gel to chek digestion efficiency. 500ng of digested DNA were Ipp over night using the Methylamp Kit from Epigentek. 50ng of digested DNA were kept as input material. Ipp and input material were amplified using the WGA2 whole genome amplification Kit from SIGMA. After Qiagen PCR cleanup, ~25ng of DNA were run in a Agilent DNA chip. Input and methyl Cytosine IPP samples were subjected to hybridization with the Roche NimbelGen rat Meth 385K Arr Del array (RN34 CpG Island). For each of the nine samples, base Cy3 labeled DNA was co-hybridized with Immunoprecipitated Cy5 labeled DNA, and raw signals were detected for all chip features on both channels. A scaled log2 ratio was calculated for each feature, and subjected to a 750pb window one-sided Kologorov-Smirnov (KS) test. The KS test determines if the probes have a more significant intensity versus the rest of the array. A negative log10 p-value is reported (e.g. 2 equals a p-value of 0.01). Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation image_aquisition hybridization nucleic_acid_extraction labeling feature_extraction Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name SAMPLE TYPE REPRODUCTIVE STAGE AGE Experimental Factor Type sample type reproductive stage age Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE38505 Comment[GEOLastUpdateDate] 2012-06-06 Comment[AEExperimentType] methylation profiling by array Comment[GEOReleaseDate] 2012-06-04 Comment[ArrayExpressSubmissionDate] 2012-06-04 SDRF File E-GEOD-38505.sdrf.txt