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Sample GSM176672 Query DataSets for GSM176672
Status Public on Mar 21, 2007
Title GFP-transduced_rep2
Sample type RNA
 
Source name GFP/MigR1 (vector-only)-transduced, live, lineage-negative, Sca1 cKit double-positive bone marrow cells
Organism Mus musculus
Characteristics C57BL/6, 5-6 weeks old
Extracted molecule total RNA
Extraction protocol Lin- Sca1+ cKit+ GFP+ (transduced) mouse bone marrow cells were sorted using a BD FACSAria directly into RLT RNA lysis buffer with 2-ME (Qiagen). Total RNA was Dnase1-treated on Rneasy mini-columns according to manufacturer's instructions (Qiagen Rneasy mini-kit). Total RNA quantity and integrity were verified using Agilent Technologies Bioanalyzer 2100.
Label biotin
Label protocol 50ng of total RNA from each sample was amplified using the RiboAmp RNA amplification kit (Arcturus). First-round aRNA was converted into double-strand cDNA again using the Arcturus RiboAmp kit. Second-round dscDNA was then biotinylated in a second amplification using the Enzo High Yield T7 transcription kit according to the manufacturer's instructions. Labeled cRNA was purified using Qiagen Rneasy mini-columns.
 
Hybridization protocol Hybridization was performed according to manufacturer's (Affymetrix) instructions
Scan protocol Scanning was performed according to manufacturer's (Affymetrix) instructions
Description Primary bone marrow mononuclear cells were harvested from 5-6 week old C57BL/6 mice (Jackson Labs, Bar Harbor, ME) and subjected to immunomagnetic negative selection using the Lineage cell depletion Kit (Miltenyi Biotech, Auburn, CA) to enrich for stem and progenitor cells (Lin- cells). To further enrich the stem cell compartment, Lin- cells underwent positive selection for the expression of Sca1 (Lin-Sca1+;LS) using the Anti-Sca-1 MicroBead Kit (Miltenyi Biotech). LS cells (6x105 in 3ml) were plated in ultra-low adhesion 6-well plates (Costar, Corning, NY) and incubated overnight at 37oC, 5% CO2 in transplant media [RPMI, 20% FCS, penicillin and streptomycin, 10 ng/ml IL-3, 20 ng/ml IL-6 (R&D Systems, Minneapolis, MN), 10 ng/ml SCF (Stem Cell technologies, Vancouver, Canada)]. Twelve hours later, 6x105 cells in 2ml of fresh transplant media were added to 6-well plates (Cellstar, GBO, NC) coated with 100 mg Retronectin (Takara, Madison WI). Independent retroviral transductions were performed on three samples using control (GFP/MigR1) retrovirus. Retroviral (GFP) supernatants (2 ml), 4 µl of 40 mg/ml polybrene and 40µl 1M Hepes were added to each well and the cells centrifuged for 90 minutes at 1400 g, 37oC. Cells were subjected to a second round of transduction 24 hours later as described above. 48 hours following the second transduction, cells were collected (kept as independent samples) and stained with PE conjugated anti-cKit (BD Bioscience), APC conjugated anti-Sca1 (Ebioscience), and biotin conjugated anti-lineage cocktail (Miltenyi Biotech). PerCP Cy5.5 conjugated Streptavidin (BD Bioscience) was added to identify differentiated cells marked with biotin conjugated antibodies. 7-AAD (Molecular Probes) was used to exclude dead cells from the sort. 25,000 transduced (GFP+), live, Lin- (7-AAD/PerCP Cy5.5-), Sca1+ cKit+ (APC-PE ++) cells were sorted directly into 600ul of RLT RNA lysis buffer (Qiagen) using a BD FACSAria cell sorter (BD Bioscience).
Data processing RMA
 
Submission date Mar 20, 2007
Last update date Aug 28, 2018
Contact name Nancy Asmussen Speck
E-mail(s) Nancy.Speck@dartmouth.edu
Phone (603) 650-1235
Fax (603) 650-1129
Organization name Dartmouth Medical School
Department Biochemistry
Lab Speck Lab
Street address 311 Vail Bldg
City Hanover
State/province NH
ZIP/Postal code 03755
Country USA
 
Platform ID GPL1261
Series (1)
GSE7324 Structural basis for recognition of SMRT/N-CoR by the MYND domain and its contribution to AML1/ETO's activity
Relations
Reanalyzed by GSE119085

Data table header descriptions
ID_REF
VALUE RMA normalized signal

Data table
ID_REF VALUE
1415670_at 3.92581462544843
1415671_at 7.11944954006911
1415672_at 8.51605926643624
1415673_at 6.19453211948744
1415674_a_at 4.62389972017154
1415675_at 4.02732117198347
1415676_a_at 7.76045683272233
1415677_at 2.20937312194178
1415678_at 6.68670713594594
1415679_at 7.4611492112088
1415680_at 7.26870992756689
1415681_at 6.12979079513164
1415682_at 1.19698247424705
1415683_at 8.3705773816559
1415684_at 2.52178756787956
1415685_at 4.32653185795303
1415686_at 4.58766969672286
1415687_a_at 4.24055833923663
1415688_at 8.2732251961176
1415689_s_at 4.40644428188831

Total number of rows: 45101

Table truncated, full table size 1270 Kbytes.




Supplementary file Size Download File type/resource
GSM176672.CEL.gz 2.4 Mb (ftp)(http) CEL
GSM176672.CHP.gz 284.9 Kb (ftp)(http) CHP
Raw data provided as supplementary file
Processed data provided as supplementary file

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