|
| Status |
Public on Dec 21, 2011 |
| Title |
VSEL_Sample2 |
| Sample type |
RNA |
| |
|
| Source name |
T7-primed cDNA libraries was synthesized using FACS sorted twenty VSELs
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Bone Marrow
cell type: VSEL
strain: C57BL/6
age: 4 weeks after birth
|
| Treatment protocol |
FACS-sorted Sca-1+Lin–CD45– VSELs, Sca-1+Lin–CD45+ HSCs, or trypsinized ESC-D3 cells were distributed into each well of a 384-well plate (Thermo Scientific) containing 4.5 ul of lysis buffer per well using a MoFlo cell sorter
|
| Growth protocol |
Bone marrow mononuclear cells (BMMNCs) were prepared from 4 weeks old mice and VSELs (Sca-1+Lin–CD45–) and HSCs (Sca-1+Lin–CD45+) was isolated by multiparameter live-cell sorting (MoFlo, Dako). Murine ESC-D3 cells (ATCC) were grown in a 0.1% gelatin-coated dish with ESC specific meida supplemented with 10 ng/ml of recombinant mouse LIF (Chemicon-Millipore).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was directly eluted from twenty cells by lysis buffer (0.5 % NP-40) according to the previous reports (Nat. Protoc. Vol2, p739-752, 2007).
|
| Label |
biotin
|
| Label protocol |
The gel-eluted T7-primed cDNA libraries were biotin-labeled using the GeneChip 3’ in vitro transcription (IVT) kit (Affymetrix), starting from ‘‘In vitro Transcription to Synthesize Labeled aRNA”.
|
| |
|
| Hybridization protocol |
Following fragmentation, 12.5 ug of cRNA were hybridized for 16 hr at 45C on GeneChip 3’ Mouse Genome 430 2.0 array (Affymetrix),according to manufacture mannual (GeneChip 3'IVT Expression Kit User Mannual, Affymetrix)
|
| Scan protocol |
GeneChips were scanned using GeneChip Scanner 3000 7G (Affymetrix) and the GeneChip Command Console 1.0 (Affymetrix)
|
| Description |
Gene expression data from PCR amplified cDNA library representign FACS sorted twenty cells
|
| Data processing |
The CEL files for 9 cell samples (3 VSEL, 3 HSC, and 3 ESC-D3) were imported into Partek software Version 6.5 (Partek Inc) and normalized using Robust Multi-Array (RMA) normalization. A one-way ANOVA was set up for the different cell types with contrasts comparing VSEL versus HSC, ESC versus HSC, and VSEL versus ESC.
|
| |
|
| Submission date |
May 13, 2011 |
| Last update date |
Dec 21, 2011 |
| Contact name |
DONG-MYUNG SHIN |
| E-mail(s) |
d0shin03@louisville.edu
|
| Phone |
502-852-3205
|
| Fax |
502-852-3032
|
| Organization name |
University of Louisville
|
| Department |
Stem Cell Institute at James Graham Brown Cancer Center
|
| Street address |
500 South, Floyd Street
|
| City |
Louisville |
| State/province |
KY |
| ZIP/Postal code |
40202 |
| Country |
USA |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE29281 |
Single Cell based genomewide gene expression analysis of murine bone-marrow derived Very Small Embryonic-Like Stem Cells (VSELs) |
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