|
| Status |
Public on Apr 30, 2014 |
| Title |
IHCC_IDH2_mutant_R172K_Collagen_Rep1 |
| Sample type |
RNA |
| |
|
| Source name |
HBs_R172K mutant IDH2_collagen-coated plates
|
| Organism |
Mus musculus |
| Characteristics |
cell type: hepatoblasts (HBs) from E14 WT mouse
genotype/variation: expressing R172K mutant IDH2
culture plate: collagen-coated plate
|
| Treatment protocol |
For IDH1 experiments, culture media was supplemented with 25 ng/mL of doxycycline (d9891, Sigma-Aldrich). For hepatocyte differentiation assays, 5e6 HB cells were cultured on uncoated 10 cm tissue culture dishes in HB cell medium for up to 5 days and isolated for gene expression analysis.
|
| Growth protocol |
HB cells were prepared from WT mice at embryonic day 14 and immortalized by plating at clonal density. HBs were maintained in HB media [DMEM/F-12 (Gibco Life Technologies, Grand Island NY) containing 10% fetal bovine serum, 1% penicillin-streptomycin, 50ng/mL epidermal growth factor, 30 ng/mL insulin-like growth factor II (PeproTech, Rocky Hill, NJ), 10 μg/mL insulin (Roche, Mannheim, Germany)] on plates coated with rat tail collagen (BD Biosciences, Bedford, MA) in a humidified atmosphere with 5% CO2 at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (1ug) was isolated using RNeasy Mini Kit (QIAGEN, Valencia, CA) and quality control performed using Bioanalyzer 2100 (Agilent Technologies).
|
| Label |
biotin
|
| Label protocol |
First strand cDNA synthesis was generated using a T7-linked oligo-dT primer, followed by second strand synthesis. An in vitro transcription reaction was performed to generate cRNA containing biotinylated UTP and CTP, which was subsequently chemically fragmented at 95°C for 35 minutes.
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| |
|
| Hybridization protocol |
The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to Affymetrix 430Av2 microarray chips overnight at 45°C. The hybridization solution is then removed. The chips are transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe.
|
| Scan protocol |
Scans were performed on Affymetrix confocal laser scanners.
|
| Description |
Cholangio_IDH2_mut_R172K_Collagen_1
|
| Data processing |
Raw expression values were normalized using Robust Multiarray Averaging (RMA).
|
| |
|
| Submission date |
Apr 23, 2014 |
| Last update date |
Apr 30, 2014 |
| Contact name |
Kenneth N Ross |
| E-mail(s) |
Kenneth.Ross@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Pediatric Oncology
|
| Street address |
450 Brookline Ave., Rm M640
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL8321 |
| Series (1) |
| GSE57002 |
Mutant IDH inhibits HNF4a to disrupt hepatocyte differentiation and promote cholangiocarcinoma. |
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