|
| 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) |
[email protected]
|
| 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 |
|
