|
| Status |
Public on Aug 04, 2011 |
| Title |
Tcf1 deficient Lineage negative cells (Mac1-Gr1-Thy1-CD25-) biological replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
murine bone marrow
|
| Organism |
Mus musculus |
| Characteristics |
strain: Tcf7-/- mice were generated by H.Clevers and backcrossed approximately 6 generations and was maintained at that mixed background
tissue: bone marrow
cell type: Lineage negative cells (Mac1-Gr1-Thy1-CD25-)
|
| Growth protocol |
Tcf1 wildtype and deficient lymphoid primed multipotent progenitors (LMPPs, Lineage marker-Sca+kit+Flt3high) were isolated by a FACSAria cell sorter and seeded onto wells containing a confluent OP9-DL4 stromal monolayer in MEMa, supplemented with 10% L-glutamate, 10%P-strep, and 20% FCS. Cytokines were added at a concentration of IL1 1ng/ml and Flt3L 5ng/ml. After four days of culture, Tcf1 wildtype and deficient lineage negative progenitors and Tcf1 wt T lineage cells (Thy1+CD25+) were isolated and visualized as DAPI-CD45.2 and then highly pure populations were cell sorted according to phenotype (Lineage negative: Mac1-Gr1-Thy1-CD25-; T lineage: Thy1+CD25+). Cells were sorted into MEMa medium and spun down and resuspended in Trizol.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufactors instructions
|
| Label |
biotin
|
| Label protocol |
10ng of total RNA was amplified using the NuGEN WTA-Ovation Pico RNA kit according to the manufactor's protocol. ST-cDNA was synthesized using NuGEN WT-Ovation Exon Module, and labeled using the NuGEN Encore Biotin Module.
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| |
|
| Hybridization protocol |
All protocols were conducted as described in the Affymetrix GeneChip Expression analysis Technical Manual (www.affymetrix.com). Biotinylated targets were heated at 99C for 5 min and hybridized for 16 h at 45C to 9 microarrays. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
GeneChips were scanned using the GeneArray Scanner 3000 7G.
|
| Description |
gene expression data from Tcf1 deficient lineage negative (CD45.2+Mac1-Gr1-Thy1-CD25-) progenitors after four days of culture on OP9-DL4 stroma
|
| Data processing |
The data were analyzed using Partek Genomics Suite, version 6.5. RMA with default settings was used to normalize data. Heat maps were drawn using the online software Matrix2png (www.bioinformatics.ubc.ca/matrix2png). For Tcf1 wildtype and deficient lineage negative progenitors, the first two representative samples are shown in the heat map.
probe group file: MoGene-1_0-st-v1.r4.pgf
meta-probeset file: MoGene-1_0-st-v1.r4.mps
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| |
|
| Submission date |
Jan 11, 2011 |
| Last update date |
Aug 04, 2011 |
| Contact name |
Avinash Bhandoola |
| E-mail(s) |
[email protected]
|
| Phone |
2155730274
|
| Organization name |
University of Pennsylvania
|
| Department |
Pathology
|
| Lab |
266 John Morgan
|
| Street address |
3620 Hamilton Walk
|
| City |
Philadelphia |
| State/province |
Philadelphia |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE26559 |
Expression data from Tcf1 deficient and Tcf1 wildtype cultured bone marrow lymphoid primed progenitors after four days on Notch ligand expressing stroma (OP9-DL4). |
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