|
| Status |
Public on Jul 04, 2013 |
| Title |
Day4_iI_Dox_3 |
| Sample type |
RNA |
| |
|
| Source name |
Day 4, ES differentiation +48hr Dox
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Ainv15 (ATCC SCRC-1029) with Dox-inducible Isl1-V5 construct
|
| Treatment protocol |
Patterning of embryoid bodies was induced by supplementing media on Day 2 with 1 µM all-trans-Retinoic acid (RA, Sigma) and 0.5 µM agonist of hedgehog signaling (SAG, Calbiochem). Doxycycline (Sigma) was added to the culture medium at 3ug/ml when required.
|
| Growth protocol |
ES cells were cultured over a layer of Mitomycin-C treated fibroblast resistant to Neomycin (Fisher) in EmbryoMax D-MEM (Fisher) supplemented with 10% ES-FBS (Invitrogen), L-Glutamine (Gibco), 0.1 mM ?-mercaptoethanol and 100 U/ml LIF. Motor neuron differentiation of ES cells was performed as previously described. Briefly, ES cells were trypsinized (Invitrogen) and seeded at 5x105 cells/ml in ANDFK medium (Advanced DMEM/F12:Neurobasal (1:1) Medium, 10% Knockout-SR, Pen/Strep, 2 mM L-Glutamine, and 0.1 mM 2-mercaptoethanol) to initiate formation of embryoid bodies (Day 0). Medium was exchanged on Day 2 and Day 5 of differentiation. Patterning of embryoid bodies was induced by supplementing media on Day 2 with 1 µM all-trans-Retinoic acid (RA, Sigma) and 0.5 µM agonist of hedgehog signaling (SAG, Calbiochem). For ChIP experiments, the same conditions were used but scaled to seed 1x107 cells on Day 0. Doxycycline (Sigma) was added to the culture medium at 3ug/ml when required.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Qiagen RNAeasy at different time points following manufacturer’s instructions.
|
| Label |
biotin
|
| Label protocol |
RNA was amplified using NuGen Applause bundle amplification and labeling kit according to manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
RNA was hybridized to Affymetrix Mouse Gene 1.0 ST microarrays.
|
| Scan protocol |
Arrays were scanned using the GeneChip Scanner 3000.
|
| Description |
Gene expression data from Day 4, ES differentiation +48hr Dox, Hb9-GFP-sorted
|
| Data processing |
Data analysis was carried out using the oneChannelGUI BioConductor package (Sanges, et al. Bioinformatics, 2007). RMA-sketch normalization was performed across all arrays. Differentially expressed genes were defined by ranking all probesets by the moderated t-statistic-derived p-value (adjusted for multiple testing using Benjamini & Hochberg’s method) and setting a threshold of p<0.001.
|
| |
|
| Submission date |
May 08, 2012 |
| Last update date |
Jul 04, 2013 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
|
| Phone |
814-865-3008
|
| Organization name |
Penn State University
|
| Department |
Biochemistry & Molecular Biology
|
| Lab |
Shaun Mahony
|
| Street address |
404 South Frear Bldg
|
| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE31456 |
Transcriptional mechanisms controlling direct motor neuron programming |
|
