|
| Status |
Public on Jun 02, 2015 |
| Title |
iHCs_Dox+RA Day 12, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
iMyo7a+ cells, FACS-sorted from day 12 embryoid bodies, treated with Dox + RA for 8 days
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic Stem Cell
cell line: iGPA-Myo7a:mVenus
age: Culture day 12
|
| Treatment protocol |
EBs were dissociated using 0.25% trypsin-EDTA (Invitrogen) in PBS and resuspended in 4% FBS in PBS. Cell sorting of iMyo7a:venus+ populations were done on a FACS Aria cell sorter (Becton Dickinson).
|
| Growth protocol |
EBs were grown in at 370C in a 5% CO2 incubator in Dulbecco's Modified Eagle’s Medium (DMEM, Invitrogen), supplemented with 10% fetal bovine serum (FBS) (ES-qualified, Invitrogen), on 60-mm bacterial-grade Petri dishes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 10e6 cells using High Pure RNA Isolation kit (Roche Diagnostics) according to manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
RNA was processed for use on Affymetrix (Santa Clara, CA, USA) Mouse Genome 2.1 ST Arrays Strip by using the Ambion WT Expression Kit (Life Technologies, CA, USA) and Affymetrix GeneChip WT Terminal Labeling Kit, according to the manufacturer’s protocols. Briefly, 100 ng of total RNA containing spiked in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) was used in a reverse transcription reaction (Ambion WT Expression Kit) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription (IVT) reaction to generate cRNA (Ambion WT Expression Kit). 15 ug of this cRNA was used for a second cycle of first-strand cDNA synthesis (Ambion WT Expression Kit). 5.5 ug of single stranded cDNA was fragmented and end-labeled (GeneChip WT Terminal Labeling Kit; Affymetrix). Size distribution of the fragmented and end-labeled cDNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
|
| |
|
| Hybridization protocol |
3.5 µg of end-labeled, fragmented cDNA was used in a 150-µl hybridization cocktail containing added hybridization controls (GeneAtlas Hybridization, Wash, and Stain Kit for WT Array Strips, Affymetrix), of which 120 ul were hybridized on array strips for 20 h at 48°C. Standard post hybridization wash and double-stain protocols (GeneAtlas Hybridization, Wash, and Stain Kit for WT Array Strips, Affymetrix) were used on an Affymetrix GeneAtlas system.
|
| Scan protocol |
Array strips were scanned on an Affymetrix GeneAtlas scanner
|
| Description |
Gene expression data from purified iHCs (iMyo7a+ cells) obtained from floating embryoid bodies treated with Dox + RA at day 4 and harvested day 12
|
| Data processing |
The 16 scanned arrays were analyzed first with Affymetrix Expression Console software using RMA to obtain expression values and for quality control. Control probe sets were removed and log2 expression values of the remaining 33708 transcripts were imported into Chipster 2.4. Differential expression was determined by empirical Bayes two-group test with Benjamini-Hochberg multiple testing correction and a p-value cut-off of 0.01.
|
| |
|
| Submission date |
Aug 12, 2014 |
| Last update date |
Jun 02, 2015 |
| Contact name |
Aida Santos da Costa |
| E-mail(s) |
aida.asc@gmail.com
|
| Organization name |
University of Edinburgh
|
| Street address |
Old College, South Bridge
|
| City |
Edinburgh |
| State/province |
Edinburgh |
| ZIP/Postal code |
EH8 9YL |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17400 |
| Series (1) |
| GSE60352 |
Analyses of iHC transcriptome profiles |
|
