|
| Status |
Public on Aug 15, 2018 |
| Title |
HL004 feeder |
| Sample type |
RNA |
| |
|
| Source name |
Human lung epithelial cell culture-method A (Patient 4)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: lung epithelial cell organoids
patient: patient 4
method: culture method A
|
| Treatment protocol |
Pneumocyte phenotype induction applied in culture-method C: The lung epithelial cells were treated with 3 µM CHIR99021, 10 ng/mL keratinocyte growth factor, 100 µM 3-isobutyl-1-methylxanthine, 100 µM 8-bromoadenosine 3’,5’-cyclic monophosphate, 25 nM dexamethasone and 10 ng/mL fibroblast growth factor-10 for three days before feeder removal.
|
| Growth protocol |
Feeder-coculture (culture-method A): The primary lung epithelial cells were cultured with with irradiated NIH-3T3 cells in an expansion medium (3:1 mixture of Ham’s F-12 nutrient mix and DMEM supplemented with 5% fetal calf serum, 0.4 µg/mL hydrocortisone , 5 µg/mL recombinant human insulin , 8.4 ng/mL cholera toxin , 24 µg/mL Adenine , and 10 ng/mL recombinant human epidermal growth factor, 9 µM Y27632 and 100 nM dibenzazepine.
Growth protocol after feeder removal used in culture-method B and C: The lung epithelial cells propagated with culture method A were passaged into a cell culture vessel after feeder removal, and cultured in DMEM supplemented with 2mM glutamine, 10mM sodium pyruvate and 10% FCS for another 72 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the TRIzol Reagent RNA preparation method (Invitrogen, Karlsruhe, Germany) using Glycogen as carrier.
|
| Label |
Cy3
|
| Label protocol |
RNA was labeled with the single-color Low Input Quick Amp Labeling Kit (Agilent Technologies) according to manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
After precipitation, purification and quantification, 0.75µg labeled samples were hybridized according to the supplier's protocol (Agilent Technologies).
|
| Scan protocol |
Scanning of microarrays was performed with 3µm resolution using a G2565CA high resolution laser microarray scanner (Agilent Technologies).
|
| Data processing |
Microarray image data were extracted and analyzed with the Image Analysis / Feature Extraction software G2567AA (Version A.10.10.1, Agilent Technologies) using default settings and the GE1_1105_Oct12 extraction protocol. The extracted single-color raw data txt files were further analyzed using R and the associated BioConductor package limma.
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| |
|
| Submission date |
Sep 06, 2016 |
| Last update date |
Aug 15, 2018 |
| Contact name |
Hans-Joachim Mollenkopf |
| E-mail(s) |
mollenkopf@mpiib-berlin.mpg.de
|
| Phone |
+49 30 28460 482
|
| Organization name |
Max-Planck-Institute for Infection Biology
|
| Lab |
Microarray/Genomics Core Facility
|
| Street address |
Charitéplatz 1
|
| City |
Berlin |
| ZIP/Postal code |
10117 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21272 |
| Series (1) |
| GSE86449 |
Gene expression profile of human lung epithelial cells with different culture methods. |
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