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Sample GSM1643002 Query DataSets for GSM1643002
Status Public on Jun 01, 2015
Title EBs_d8_Hopx-Null + Wnt_Inhibitor vs Hopx-Het_rep3
Sample type RNA
 
Channel 1
Source name d8 mouse embryoid bodies
Organism Mus musculus
Characteristics tissue: d8 mouse embryoid bodies
genotype: Hopx-/- + Wnt Inibitor EBs
Treatment protocol Cardiac differentiation was adapted from published protocols. Briefly, ESCs were cultured and maintained on a feeder layer of mitotically inactivated MEFs in DMEM with 15% FBS (Fisher Scientific) and ESGRO leukemia inhibitory factor. Differentiation through hanging droplets method was initiated following ESC dissociation and suspension at 5 x 104 cells/ml in DMEM with 10% FBS (Atlanta Biologicals) without LIF in 20µl drops. Two days after droplets formation, EBs were transferred in suspension onto poly-HEMA coated dishes. After another two days, EBs were plated on gelatin coated dishes in cardiac differentiation media [StemPro-34 SF medium (Invitrogen) supplemented with 5 ng/ml VEGF (R&D systems), 10 ng/ml bFGF (R&D systems), 12.5 ng/ml FGF10 (R&D systems), 1mM Ascorbic Acid (Sigma), 2mM Glutamax (Invitrogen)], and XAV939 as indicated. RNA was isolated from day 8 cultures using Trizol (Life Techonologies) according to manufacturer's instructions.
Growth protocol Embryonic stem cells were derived from Hop+/- and Hopx-/- blastocysts using standard protocols. Briefly, blastocysts were collected at E3.5 and cultured on STO feeder cells in standard ESC media +LIF + 50µM MEK1 inhibitor (Cell Signaling) for 7 days until the blastocyst hatches and forms colony. Individual colonies were subcultured for approximately 1 week when MEK1 inhibitor is removed and cells passaged as a normal ESC line.
Extracted molecule total RNA
Extraction protocol 200ng of total RNA were primed with 7 µl of 100 µM T7 DNA primer at 65°C for 10 min, then reversed transcribed at 40°C for 2 h in the presence of 1.2ul of Affinity Script BlockMix (Agilent) and 100 µM each dATP, dTTP, dGTP
Label Cy5
Label protocol cDNA labeled at 40°C for 2 hours with 25 µM dCTP or 25 µM Cy3-label in the prescence of T7 RNA Polymerase (Agilent)
 
Channel 2
Source name d8 mouse embryoid bodies
Organism Mus musculus
Characteristics tissue: d8 mouse embryoid bodies
genotype: Hopx+/- EBs
Treatment protocol Cardiac differentiation was adapted from published protocols. Briefly, ESCs were cultured and maintained on a feeder layer of mitotically inactivated MEFs in DMEM with 15% FBS (Fisher Scientific) and ESGRO leukemia inhibitory factor. Differentiation through hanging droplets method was initiated following ESC dissociation and suspension at 5 x 104 cells/ml in DMEM with 10% FBS (Atlanta Biologicals) without LIF in 20µl drops. Two days after droplets formation, EBs were transferred in suspension onto poly-HEMA coated dishes. After another two days, EBs were plated on gelatin coated dishes in cardiac differentiation media [StemPro-34 SF medium (Invitrogen) supplemented with 5 ng/ml VEGF (R&D systems), 10 ng/ml bFGF (R&D systems), 12.5 ng/ml FGF10 (R&D systems), 1mM Ascorbic Acid (Sigma), 2mM Glutamax (Invitrogen)], and XAV939 as indicated. RNA was isolated from day 8 cultures using Trizol (Life Techonologies) according to manufacturer's instructions.
Growth protocol Embryonic stem cells were derived from Hop+/- and Hopx-/- blastocysts using standard protocols. Briefly, blastocysts were collected at E3.5 and cultured on STO feeder cells in standard ESC media +LIF + 50µM MEK1 inhibitor (Cell Signaling) for 7 days until the blastocyst hatches and forms colony. Individual colonies were subcultured for approximately 1 week when MEK1 inhibitor is removed and cells passaged as a normal ESC line.
Extracted molecule total RNA
Extraction protocol 200ng of total RNA were primed with 7 µl of 100 µM T7 DNA primer at 65°C for 10 min, then reversed transcribed at 40°C for 2 h in the presence of 1.2ul of Affinity Script BlockMix (Agilent) and 100 µM each dATP, dTTP, dGTP
Label Cy3
Label protocol cDNA labeled at 40°C for 2 hours with 25 µM dCTP or 25 µM Cy3-label in the prescence of T7 RNA Polymerase (Agilent)
 
 
Hybridization protocol Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed in Agilent GE wash buffers 1-3.
Scan protocol Scanned on an Agilent G2565AA scanner. Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
Description Replicate 3 of 3. Null + Wnt Inibitor vs hererozygous EBs
Data processing Agilent Feature Extraction Software (v 8.5.1.1) was used for background subtraction and LOWESS normalization.
 
Submission date Mar 25, 2015
Last update date Jun 01, 2015
Contact name Kyoung Jae Won
E-mail(s) wonk@mail.med.upenn.edu
Organization name University of Pennsylvania
Department Genetics
Lab WonLab
Street address 3400 Civic Center Blvd
City Philadelphia
ZIP/Postal code 19104
Country USA
 
Platform ID GPL10333
Series (2)
GSE67252 Role of Hopx in myogenesis and commitment [HopxKO+Wnt]
GSE67254 Role of Hopx in myogenesis and commitment

Data table header descriptions
ID_REF
VALUE Normalized log10 ratio (Cy5/Cy3) representing experimental/control

Data table
ID_REF VALUE
12 0.183
13 -0.053
14 0.159
15 -0.071
16 0.244
17 -0.179
18 0.134
19 0.013
20 -1.042
21 0.007
22 0.363
23 -1.325
24 -0.032
25 -0.839
26 0.516
27 0.499
28 0.747
30 -2.251
31 0.502
32 0.049

Total number of rows: 43173

Table truncated, full table size 508 Kbytes.




Supplementary file Size Download File type/resource
GSM1643002_252665516781_2.txt.gz 4.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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