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Sample GSM381324 Query DataSets for GSM381324
Status Public on Jul 13, 2010
Title Mesodermal progenitors with inducible Notch1; OHT induced; CHX treated; replicate 1
Sample type RNA
 
Source name NERT mesodermal progenitors, w/ OHT 4h, w/ CHX 4 h
Organism Mus musculus
Characteristics strain: EB5
cell type: mesodermal progenitor
nert: +
condition: Collagen+FCS
oht: +
chx: +
days differentiation: 4
Treatment protocol For induction of Notch1 signaling, 1 µM tamoxifen (OHT) was added to the culture medium for 4 h. To block protein synthesis, 50 µg/ml cycloheximide (CHX) was added for 4 h in parallel to induction.
Growth protocol Culture of undifferentiated ESC and the generation of mesodermal cells was performed as described (Schroeder et al. 2003, http://www.pnas.org/content/100/7/4018.long). Briefly, EB5 cells carring a tamoxifen inducible NERT construct or an empty control were kept under self-renewal conditions. For the culture of cells in conditions favoring ectodermal or mesodermal differentiation, ESC grown on gelatine were cultured for 4 h in GMEM, 10% Knockout Serum Replacement, 1% non essential amino acids, 1 mM sodium pyruvate and 100 µM 2-mercaptoethanol (ES to ectoderm), or in Knockout-DMEM, 10% Knockout Serum Replacement, 1% pre-tested FCS, 1% non essential amino acids and 100 µM 2-mercaptoethanol (ES to mesoderm). Mesodermal progenitors were generated by transferation of EB5 cells into mesodermal differentiation medium on Collagen IV coated dishes and kept for 4 d. Successful differentiation was monitored by generation of Flk1+ cells via FACS analysis.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using RNeasy Midi kit (Qiagen, Hilden, Germany) with DNAse digestion using the maufacturer's buffers and protocols. RNA quality was assessed by Agilent 2100 Bioanalyzer System.
Label biotin
Label protocol Probe preparation was with 5 microgram RNA using the Affymetrix One-Cycle Target Labeling Kit according to the manufacturer's instructions. Labeled RNA was fragmented by heating to 95°C for 35 minutes in 200 mM TRIS (pH 8.1), 500 mM KOAc, 150 mM MgOAc buffer.
 
Hybridization protocol Affymetrix arrays were prehybridized with 200 microliter MES hybridization buffer for 10 minutes at 45°C with rotation (60 U/min) in an Affymetrix Hybridization Oven 640. After removing the prehybridization solution, 15 microgram of labeled cRNA in 200 microliter MES hybridization buffer were applied to the array leaving a small air bubble. Arrays were incubated overnight at 45°C with rotation (60U/min) in the hybridization chamber.
Scan protocol Arrays were scanned using Affymetrix GeneChip Scanner 3000.
Description Gene expression data from mesodermal progenitors
Data processing The data were analyzed with dChip software (http://www.dchip.org/; Built date:Aug 21 2008) using dChip default analysis settings and were normalized to an array with median overall intensity ('Con_M+' = ‘Control Mesoderm +OHT’) using the Invariant Set Normalization method.
 
Submission date Mar 17, 2009
Last update date Jul 13, 2010
Contact name Ralf Schwanbeck
E-mail(s) [email protected]
Organization name University of Kiel
Department Biochemistry
Street address Otto-Hahn-Platz 9
City Kiel
ZIP/Postal code 24118
Country Germany
 
Platform ID GPL1261
Series (1)
GSE15268 Cell-context dependent Notch target genes

Data table header descriptions
ID_REF
VALUE dChip signal intensity
ABS_CALL

Data table
ID_REF VALUE ABS_CALL
1415670_at 498.58 P
1415671_at 932.46 P
1415672_at 2808.44 P
1415673_at 582.31 P
1415674_a_at 919.48 P
1415675_at 534.83 P
1415676_a_at 1591.75 P
1415677_at 63.83 P
1415678_at 1042.01 P
1415679_at 1222.41 P
1415680_at 2904.8 P
1415681_at 1181.97 P
1415682_at 540.46 P
1415683_at 4472.09 P
1415684_at 306.97 P
1415685_at 538.52 P
1415686_at 990.5 P
1415687_a_at 1059.47 P
1415688_at 2538.1 P
1415689_s_at 288.05 P

Total number of rows: 45037

Table truncated, full table size 859 Kbytes.




Supplementary file Size Download File type/resource
GSM381324.CEL.gz 3.8 Mb (ftp)(http) CEL
Processed data included within Sample table

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