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Sample GSM1613970 Query DataSets for GSM1613970
Status Public on Apr 15, 2015
Title iPSC wild-type T24 3
Sample type RNA
 
Source name wild-type skin fibroblasts
Organism Homo sapiens
Characteristics cell line: iPSC
Stage: T24
group: iPSC wild-type
Treatment protocol Aside from differentiation and sorting as described above, cells were not treated in any specific manner prior to DNA/RNA extraction.
Growth protocol iPSC and ESC lines were maintained in “hiPSC Media” composed of DMEM/F12 (Sigma-Aldrich, www.sigma.com) with 20% KnockOut Serum Replacement (Invitrogen, www.invitrogen.com), 1mM nonanimal L-glutamine (Sigma-Aldrich), 0.1mM Β-mercaptoethanol, and 10 ng/ml FGF2 (R&D Systems, Minneapolis, MN) on 0.1% gelatin (Sigma-Aldrich) coated plates preseeded with mitomycin C-inactivated or irradiated mouse embryonic fibroblast (MEF) feeder cells. Cells were maintained in a 5% CO2 air environment. For endodermal differentiation, cells were passed onto matrigel-coated dishes at 80% confluency. On the following day, designated “T0”, differentiation was induced by culture in media containing growth factors listed below. From T0-T6, differentiation media included 2mM l-glutamine, and 4.5x10-4 M monothioglycerol (MTG). Cells were grown in T0 media, consisting of RPMI-based serum-free medium with Chir 99021 (2ug/ml) and Activin A (100 ng/ml), for one day. On days “T1-2”, medium was changed to RPMI with BMP4 (0.5 ng/ml), FGF2 (10ng/ml), Activin A (100 ng/ml), and VEGF (10 ng/ml). On days “T3-4”, cells were cultured in SFD media(Gouon-Evans et al., 2006) with BMP4 (0.5 ng/ml), FGF2 (10ng/ml), Activin A (100 ng/ml), and VEGF (10 ng/ml). For hepatic differentiation, PSCs were differentiated as monolayer cultures as outlined above to generate definitive endoderm and then further differentiated for an additional 3 weeks in SFD-based media with ascorbic acid (50mcg/ml), monothioglycerol (4.5x10-4 M ), and the following supplements: T7-12: BMP4 (50 ng/ml), FGF2 (10 ng/ml), VEGF (10 ng/ml), EGF (10ng/ml), TGFa (20 ng/ml), HGF (100 ng/ml), and 0.1 uM Dexamethasone; T13-18: FGF2 (10 ng/ml), VEGF (10 ng/ml), EGF (10 ng/ml), HGF (100 ng/ml), Oncostatin M (20 ng/ml), Vitamin K (6 ug/ml), 1.5 uM gamma secretase inhibitor, 0.1 uM Dexamethasone, and 1% DMSO; T19-24: HGF (100 ng/ml), Oncostatin M (20 ng/ml), Vitamin K (6 ug/ml), and 0.1 uM Dexamethasone. Differentiating human PSCs were maintained in a 5% CO2, 5% O2, 90% N2 environment.
Extracted molecule total RNA
Extraction protocol Total RNA and miRNA were isolated from cells using an miRNeasy kit (Qiagen) with the optional column RNAse-free DNase treatment, according to the manufacturer’s instructions. 200 nanograms to one microgram of RNA was reverse transcribed into cDNA using random hexamers with Superscript III Reverse Transcriptase (Invitrogen). RNA was extracted at each stage, biotin labeled, and hybridized to either Affymetrix GeneChip Human Gene 1.0 ST or miRNA 2.0 arrays. For methylation analysis, gDNA was extracted from the same 27 samples before undergoing bisulfite conversion. Bisulfite converted DNA was then amplified and purified prior to overnight hybridization to Illumina’s Infinium HumanMethylation 450 BeadChips. Next day staining of hybridized arrays produced methylation-dependent differential fluorescence that was detected via an Illumina iScan array scanner.
Label biotin
Label protocol Biotin labeling was performed using the Ambion WT Expression Kit (Life Technologies, Grand Island, NY) according to the manufacturer's protocol, followed by the GeneChip WT Terminal Labeling and Controls Kit (Affymetrix, Santa Clara, CA).
 
Hybridization protocol The labeled, fragmented DNA was hybridized to the Affymetrix GeneChip Human Gene 1.0 ST Array for 18 hours in a GeneChip Hybridization oven 640 at 45oC with rotation (60 rpm). The hybridized samples were washed and stained using an Affymetrix fluidics station 450.
Scan protocol After staining, microarrays were immediately scanned using an Affymetrix GeneArray Scanner 3000 7G Plus.
Description Sample name: HRII3 T24
Gene expression data from wild-type skin fibroblasts profiled at T24.
Data processing The data were analyzed with the Robust Multiarray Average (RMA) using the Affymetrix Expression Console.
 
Submission date Feb 19, 2015
Last update date Apr 16, 2015
Contact name Adam C Gower
E-mail(s) agower@bu.edu
Phone 617-358-7138
Organization name Boston University School of Medicine
Department Department of Medicine
Lab Division of Computational Biomedicine
Street address 72 East Concord Street, E632
City Boston
State/province MA
ZIP/Postal code 02118
Country USA
 
Platform ID GPL6244
Series (2)
GSE66076 Emergence of a developmental stage-dependent human liver disease signature demonstrated by directed differentiation of alpha-1 antitrypsin deficient iPS cells [HuGene-1_0-st]
GSE66078 Emergence of a developmental stage-dependent human liver disease signature demonstrated by directed differentiation of alpha-1 antitrypsin deficient iPS cells

Data table header descriptions
ID_REF
VALUE log2-transformed RMA signal intensity

Data table
ID_REF VALUE
7896738 3.281451
7896740 3.441759
7896742 8.235366
7896744 5.28442
7896746 9.654428
7896748 9.406874
7896750 6.22201
7896752 10.30138
7896754 7.581694
7896756 4.925167
7896759 6.30278
7896761 6.233428
7896779 6.603643
7896798 6.9282
7896817 6.831896
7896822 8.627121
7896859 5.825219
7896861 3.186462
7896863 5.636741
7896865 6.635563

Total number of rows: 29198

Table truncated, full table size 481 Kbytes.




Supplementary file Size Download File type/resource
GSM1613970_DK_E12_56.CEL.gz 3.8 Mb (ftp)(http) CEL
Processed data included within Sample table

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