|
| Status |
Public on Nov 21, 2016 |
| Title |
Ebf1(-/-)::Ebf1-H240A-Rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Ebf1−/− c-Kit+ fetal liver cells, pmys-EBF1-H240A-IRES-GFP,Replicate3
|
| Organism |
Mus musculus |
| Characteristics |
tissue: fetal liver
genotype: Ebf1-/-
cell marker: c-Kit+
background strain: C57Bl/6
transductant: Ebf1-H240A
|
| Treatment protocol |
The cells were transduced with pMys-IRES-GFP (vector), pMys-EBF1wt-IRES-GFP or pMys-EBF1-H240A-IRES-GFP retroviruses. 36h after transduction, GFP+ cells were sorted and RNA was prepared.
|
| Growth protocol |
Single cell suspensions of freshly isolated Ebf1−/− fetal liver cells were stained with biotinylated c-Kit and c-Kit positive cells were enriched on an AutoMACS separator and subsequently 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).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was extracted using RNeasy Mini Kit according to the protocol recommended by the manufacturer.
|
| 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.
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| |
|
| Hybridization protocol |
1,65 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 55µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Mouse Genome Microarray 4x44K (14868) 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 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after pmys-EBF1-H240A_rep3-IRES-GFP transduction
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_F_20131202) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Oct 05, 2016 |
| Last update date |
Nov 21, 2016 |
| 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) |
| GSE87634 |
Gene expression profile of EBF1-wt & EBF1-H240A |
| GSE87637 |
Interaction of CNOT3 with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis |
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