|
| Status |
Public on Sep 12, 2012 |
| Title |
H14, DFNB |
| Sample type |
RNA |
| |
|
| Source name |
hESCs differentiating with basal DFNB medium
|
| Organism |
Homo sapiens |
| Characteristics |
time: Day 14
cell line: H14
treatment: DFNB
|
| Treatment protocol |
Undifferentiated hESCs were dissociated with Trypsin -EDTA (Sigma) and the cell suspension passed through a 100-micrometer cell strainer (BD Labware). Cells were plated onto laminin-coated plastic and incubated with DFNB medium (DMEM/high glucose: F12 mixed as 1:1, with N2 and B27) supplemented with FGF3 (50ng/ml) and FGF10 (50ng/ml) or with DFNB alone, without FGFs. Cells were allowed to differentiate for 14 days.
|
| Growth protocol |
hESC lines H14, Shef1 and Shef3 were maintained on inactivated mouse embryonic fibroblast (MEF) feeder cells in medium comprised of knockout Dulbecco’s modified Eagle’s medium supplemented with 20% knockout serum replacement (KOSR), 1% nonessential amino acids, 2mM L-glutamine, (all from Invitrogen), 0.1mM ß-mercaptoethanol (Sigma), and 4ng/ml of basic fibroblast growth factor (bFGF; R&D systems).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNAeasy Kit (Qiagen) following the manufacturer's instructions
|
| Label |
Biotin
|
| Label protocol |
200ng of total RNA was amplified using the 3' IVT method and biotinylation of antisense RNA carried out according to Affymetrix protocol
|
| |
|
| Hybridization protocol |
12.5µg of cRNA molecules were heat fragmented and applied to the Human Genome U133 plus 2 GeneChip in a hybridisation solution according to the Affymetrix protocol. Hybridisation took place overnight in a rotating hybridisation platform at 45°C for 16 hours. Post hybridisation stringency washing was done using the Affymetrix fluidics station again following the protocol outlined in the Affymetrix instructions
|
| Scan protocol |
the GeneChips were scanned in the Affymetrix GCS3000 scanner and the resultant image processed using the GCOS software package to prepare the .CEL files
|
| Data processing |
Normalization and initial analysis were done using puma (www.bioconductor.org) that estimates gene expression levels using probabilistic models
|
| |
|
| Submission date |
Mar 23, 2012 |
| Last update date |
Sep 12, 2012 |
| Contact name |
Marcelo Nicolas Rivolta |
| E-mail(s) |
m.n.rivolta@shef.ac.uk
|
| Phone |
44-114-222 2385
|
| Fax |
44-114-222 2360
|
| Organization name |
University of Sheffield
|
| Department |
Biomedical Science
|
| Street address |
Western Bank
|
| City |
Sheffield |
| State/province |
Yorkshire |
| ZIP/Postal code |
S10 2TN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE36754 |
Gene expression of differentiating hESCs into otic progenitors |
|
