|
| Status |
Public on Oct 19, 2020 |
| Title |
dHet-proB-Rep2 (array) |
| Sample type |
RNA |
| |
|
| Source name |
Ebf1+/− Pax5+/- dHet fetal liver proB cells,Replicate2
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Ebf1+/− Pax5+/- Rag2-/- proB fetal liver
cell type: dHet proB cells
genotype: Ebf1+/− Pax5+/- Rag2-/-
|
| Growth protocol |
dHet leukemic cells were derived from the lymph node of Ebf1+/-Pax5+/- leukemic mice. dHet proB cells were derived by culturing the single-cell suspensions of freshly isolated c-Kit positive fetal liver cells were plated on OP9 feeder cells in OptiMEM medium, supplemented with 4% FCS, 1% PSG, 50 μM β-mercaptoethanol, 5 ng/ml interleukin 7 (IL-7), 10 ng/ml Flt3 ligand (Flt3L) and 10 ng/ml stem cell factor (SCF). wt proB (FrBC) cells were sorted from the control mice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was prepared with the RNeasy Mini Kit following the manufacturer's recommendations.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the Quick Amp Labeling Kit (One-Color) and RNA Spike-In Kit, according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in dHet proB cells
dHet-proB-R2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background-subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Sep 28, 2020 |
| Last update date |
Oct 19, 2020 |
| Contact name |
Senthilkumar Ramamoorthy |
| E-mail(s) |
senthilkumar@ie-freiburg.mpg.de
|
| Phone |
00497615108731
|
| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
|
| Department |
Cellular & Molecular Immunology
|
| Lab |
Laboratory Rudolf Grosschedl
|
| Street address |
Stübeweg 51
|
| City |
Freiburg |
| ZIP/Postal code |
D-79108 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE158645 |
Microarray analysis to unravel molecular networks driving leukemia in Ebf1+/- Pax5+/- (dHet) B-ALL mice |
| GSE158673 |
EBF1 and Pax5 safeguard leukemic transformation by limiting IL-7 signaling, Myc expression and folate metabolism |
|
