|
| Status |
Public on Mar 21, 2015 |
| Title |
GMP cells from FLT3/ITD mouse bone marrow |
| Sample type |
RNA |
| |
|
| Source name |
FLT3/ITD GMP
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
genotype/variation: FLT3/ITD
tissue: bone marrow
cell type: GMP cells
|
| Treatment protocol |
Mouse bone marrow cells were harvested by flushing the tibia and femur bones with PBS, stained with antibodies and sorted for KSL and GMP cells.
|
| Growth protocol |
C57BL/6 wild-type and transgenic FLT3/ITD and SmoM2; FLT3/ITD mice were bred in the animal facility. Poly(I:C) was administered via intraperitoneal injections upon weaning.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from sorted mouse bone marrow cells (KSL and GMP populations) was extracted using the Qiagen Rneasy Micro kit and processed using the RNA amplification protocol described by Affymetrix (Affymetrix 3’ IVT Express Manual).
|
| Label |
Biotin
|
| Label protocol |
100 nanograms of total RNA were used to synthesize first strand cDNA using oligonucleotide probes with 24 oligo-dT plus T7 promoter as primer and the SuperScript Choice System (Invitrogen, Carlsbad, California). Following the double stranded cDNA synthesis, biotinilated anti-sense cRNA was generated through in vitro transcription using the T7 RNA polymerase.
|
| |
|
| Hybridization protocol |
15 ug of the biotinilated cRNA were fragmented at 94 C for 35 min (100mM Tris-acetate, pH 8.2, 500mM KOAc, 150mM MgOAC), and 10ug of total fragmented cRNA were hybridized to the Affymetrix GeneChip arrays for 16hr at 45 C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was then used to wash and stain the Chips, removing the non-hybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinilated cRNA. The staining was then amplified using goat IgG as blocking reagent and biotinilated anti-streptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate.
|
| Scan protocol |
Fluorescence was detected using the Affymetrix 3000 GeneArray Scanner with G7 upgrade and image analysis of each GeneChip was done through the Affymetrix GeneChip Command Console 3.4.1 (AGCC) software from Affymetrix, using the standard default settings.
|
| Description |
Gene expression data from GMP cells of FLT3/ITD mouse bone marrow
|
| Data processing |
Affymetrix Expression Console (v 1.2.0.20) was used to analyze the data to generate normalized expression values.
affymetrix-algorithm-name = ExpressionStat
affymetrix-algorithm-version = 5.0
program-name = Expression Console
program-id = 1.2.0.20
|
| |
|
| Submission date |
Mar 20, 2015 |
| Last update date |
Mar 23, 2015 |
| Contact name |
Yiting Lim |
| Organization name |
Johns Hopkins University School of Medicine
|
| Street address |
1650 Orleans Street
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21287 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE67134 |
Expression data from KSL and GMP cells of FLT3/ITD and FLT3/ITD-SmoM2 mice |
|
