|
| Status |
Public on Jul 01, 2014 |
| Title |
Day 4 EB_GemKD_control_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
day 4 embryoid bodies, GemKD, no dox control
|
| Organism |
Mus musculus |
| Characteristics |
background/strain: 129P2/OlaHsd
es cell line: A2lox
es cell line type: Geminin knockdown
cell type: embryoid bodies
differentiation day: 4
dox addition: no Dox
|
| Treatment protocol |
Embryoid bodies from the Gem knockdown line were collected on day 4. Embryoid bodies from the Gem overexpression line were collected on days 4 or 5.
|
| Growth protocol |
ES cells were differentiated into embryoid bodies for 4 or 5 days. ES cells were maintained on irradiated MEFs in DMEM plus 15% fetal bovine serum (HyClone), 1mM sodium pyruvate, 0.1mM non-essential amino acids (Gibco), 2mM Glutamine, 1000U/mL LIF (Chemicon) and 100μM 2-mercaptoethanol (Sigma). For EB formation, ES cells were feeder subtracted and plated at 6,000/ml on 10 cm petri dishes treated with 5% polyheme to prevent cell adhesion and were differentiated in IMDM with 15% FCS, 1% L-glutamine, 50µg/ml ascorbic acid and 4.5X10-4 M MTG. Dox is added at 0.5 µg/ml to induce overexpression of Gem or miRNAs targeting endogenous Gem transcripts.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepped with the Qiagen RNeasy kit.
|
| Label |
biotin
|
| Label protocol |
1 microgram of RNA was prepared by the Whole Transcript Sense Target Labeling (Affymetrix) standard protocol to add biotin label.
|
| |
|
| Hybridization protocol |
RNA was hybridized to Affymetrix Mouse Gene 1.0 ST microarrays and washed and stained with the GeneChip Fluidics Station 450 (Affymetrix) according to standard protocols.
|
| Scan protocol |
Array image acquisition was done with GeneChip Scanner 3000 7G (Affymetrix) and image processing with Affymetrix GeneChip Command Console (AGCC) software.
|
| Description |
Day 4 EB, rep 2, control
|
| Data processing |
Raw CEL and DAT files were analyzed with dChip software (http://biosun1.harvard.edu/complab/dchip/) after normalization to exclude probe sets that did not meet the following preliminary cutoff values: 1) expression, represented by model-based expression indices (MBEI), was changed more than 1.2-fold in treated versus untreated samples (E/B >1.2) groups; 2) MBEI differences were larger than 30 (E-B > 30); and 3) expression was claimed as present (P) in at least one sample in each experiment. For each sample type, the entire experiment was repeated three times. Probe sets were considered putatively Geminin-regulated if they met microarray cutoff values in at least 2 of the 3 experiments.
|
| |
|
| Submission date |
Jul 26, 2012 |
| Last update date |
Jul 01, 2014 |
| Contact name |
Kristen L Kroll |
| E-mail(s) |
kkroll@wustl.edu
|
| Organization name |
Washington University School of Medicine
|
| Department |
Developmental Biology
|
| Lab |
Kristen Kroll
|
| Street address |
320 McDonnell Sciences/660 S. Euclid Ave.
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE39676 |
Geminin represses mesendoderm cell fate acquisition in embryoid bodies |
|
