Comment[ArrayExpressAccession] E-GEOD-51748 MAGE-TAB Version 1.1 Public Release Date 2014-01-01 Investigation Title Comparison of the molecular profiles of human embryonic and isogenic induced pluripotent stem cells. Comment[Submitted Name] Comparison of the molecular profiles of human embryonic and isogenic induced pluripotent stem cells. Experiment Description This SuperSeries is composed of the SubSeries listed below. Refer to individual Series 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 Johnson Person First Name Kory Person Mid Initials R Person Email johnsonko@ninds.nih.gov Person Affiliation NINDS/NIH Person Phone 301-402-1956 Person Address DIR IT & Bioinformatics, NINDS/NIH, 10/5S223, 9000 Rockville Pike, Bethesda, MD, USA Person Roles submitter Protocol Name P-GSE51748-12 P-GSE51748-2 P-GSE51748-4 P-GSE51748-11 P-GSE51748-10 P-GSE51748-9 P-GSE51748-13 P-GSE51748-5 P-GSE51748-1 P-GSE51748-6 P-GSE51748-3 P-GSE51748-7 P-GSE51748-8 P-GSE51748-21 P-GSE51748-17 P-GSE51748-22 P-GSE51748-18 P-GSE51748-14 P-GSE51748-15 P-GSE51748-20 P-GSE51748-16 P-GSE51748-19 P-GSE51748-23 Protocol Description IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_11_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_1_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_3_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_10_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_9_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_8_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_12_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_4_DetectionPval = The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-1100-Jul11 and Grid: 014868_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. ID_REF = VALUE = gProcessedSignal IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_5_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_2_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_6_DetectionPval = IlluminaM-bM-^@M-^Ys GenomeStudio V2011.1 Methylation Module v1.9.0 was used. ID_REF = VALUE = Average Beta SAMPLE_7_DetectionPval = Cy5 and Cy3 Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer. Bisulphite converted DNA was amplified, fragmented and hybridized to Illumina Infinium Human Methylation27 Beadchip using standard Illumina protocol. 1.65 ug of Cy3-labelled cRNA was fragmented at 60M-0C for 30 minutes in a reaction volume of 55ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturerM-bM-^@M-^Ys instructions. On completion of the fragmentation reaction, 55ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112F) for 17 hours at 65M-0C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37M-0C GE Wash buffer 2 (Agilent). NA All culture reagents were acquired from Invitrogen unless stated otherwise. Standard culture conditions of 37oC, 5% CO2 and 95% humidity were maintained for all cells. Human ES cells (hESCs) were cultured on a feeder-layer of irradiated CF1 mouse embryonic fibroblasts (MEFs) in DMEM:F12 (Cat# 11330-032) containing 20% Knockout Serum Replacement (KSR)(Cat# 10828-028), 1mM glutamine (Cat# 25030-081), 0.1mM M-oM-^AM-"-mercaptoethanol (M-oM-^AM-"-ME; Sigma), 1x non-essential amino acids (NEAA; Cat# 11140-050) and 4ng/ml bFGF (R&D Systems)(Cat# 233-FB). Fibroblasts were cultured in DMEM (Cat# 11965-092) containing 10% fetal bovine serum (FBS) (Gemini Bio-products), 2mM glutamine and 1x NEAA. Fibroblasts were irradiated with ~6500 rads using a Faxitron RX650 X-irradiator. They were subsequently plated on Falcon 6-well tissue culture dishes, coated with 0.1% gelatin, at a density of 0.1875 x 106/well. hESCs were plated in small clumps the following day, medium was exchanged every day and colonies were passaged by collagenase treatment every 3-4 days. Embryoid bodies were cultured in fibroblast medium (FBS; EB_mesend) or in hESC medium without bFGF (KSR:EB_ecto) in 60mm Corning Low Attachment dishes for a total of 8 days. Media were changed by sedimentation every 2 days. Genomic DNA was extracted using the Promega Wizard Genomic DNA Purification Kit according to the manufacturerM-bM-^@M-^Ys instructions. RNA was extracted using a modification of the basic Trizol (Invitrogen) protocol. Briefly, 1ml of Trizol was added to sedimented colonies or EBs and triturated to dissociate the cells. At this point the lysates were stored at -80oC until all samples for that cell line were collected. Upon thaw, lysates were incubated at room temperature for 10 mins, mixed with 200M-oM-^AM--l chloroform and centrifuged in a Phase-Lock Gel (Heavy) Eppendorf tube (Qiagen). RNA was precipitated from the aqueous phase by the addition of 250M-oM-^AM--l of isopropanol and 250M-oM-^AM--l of a high salt buffer (0.8 M sodium citrate and 1.2 M NaCl) followed by centrifugation. The RNA pellet was washed twice with 75% ethanol, dried and resuspended in nuclease-free water. RNA was DNase treated for 20 mins and the DNAse removed using AmbionM-bM-^@M-^Ys DNA-Free kit. Concentration was determined using a NanoDrop ND-1000 UV-VIS Spectrophotometer. Slides were scanned immediately after washing on a Surescan Microarray Scanner G2600D using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR high 100%, XDR low 10%). Arrays were imaged using IlluminaM-bM-^@M-^Ys iScan BeadArray Scanner in conjunction with manufacturer recommended settings. Protocol Type normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol normalization data transformation protocol labelling protocol labelling protocol hybridization protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol nucleic acid extraction protocol array scanning protocol array scanning protocol Experimental Factor Name PASSAGE STATE CELL TYPE CLASS CONDITION LINE Experimental Factor Type passage state cell type class condition line Comment[SecondaryAccession] GSE51748 Comment[GEOReleaseDate] 2014-01-01 Comment[ArrayExpressSubmissionDate] 2013-10-26 Comment[GEOLastUpdateDate] 2014-01-02 Comment[AEExperimentType] transcription profiling by array Comment[AEExperimentType] methylation profiling by array SDRF File E-GEOD-51748.sdrf.txt