NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1001373 Query DataSets for GSM1001373
Status Public on Dec 31, 2012
Title unstimulated vs unstimulated 73_FNV_Normal 1
Sample type RNA
 
Channel 1
Source name DN T-cells
Organism Cercocebus atys
Characteristics cd4 status: Normal
treatment: Unstimulated
cell type: Double Negative (CD4-/CD8-) T-cells
infection: SIV
Treatment protocol 1 million purified T cells were stimulated with anti-CD3 (3mg/ml) and anti-CD28 (1mg/ml) in 200ul of complete RPMI at 37oC for 4 hrs.
Growth protocol Pure population of double negative and CD4 T cells were isolated by a step-wise magnetic bead isolation (Miltenyi) using bead specific for non-human primate antigens. Briefly, we stained PBMC with CD4-Microbead to isolate CD4 cells and treated the flow-through with CD8-PE and CD16-PE followed by anti-PE Microbeads for CD8 and CD16 depletion. This CD4-CD8-CD16- flow-through was then positive selected for CD3+ cells with CD3-Biotin and anti-biotin Microbead to obtain, CD3+CD4-CD8- (double negative T cells). Purity was checked after each isolation and further experiments were carried out only with >98% pure populations. Cells were rested overnight prior to functional analyses.
Extracted molecule total RNA
Extraction protocol Cells were collected post-stimulation and immediately lysed in RLT buffer (Qiagen) for RNA extraction. RNA was isolated using RNeasy mini kit (Qiagen) and genomic DNA was removed on-column.
Label Cy3
Label protocol cRNA was synthesized from total RNA and Cy3/Cy5 labeled using the Agilent Low Input Quick Amp Labeling procedure, as per the manufacturer’s instructions.
 
Channel 2
Source name DN T-cells
Organism Cercocebus atys
Characteristics cd4 status: Normal
treatment: Unstimulated
cell type: Double Negative (CD4-/CD8-) T-cells
infection: SIV
Treatment protocol 1 million purified T cells were stimulated with anti-CD3 (3mg/ml) and anti-CD28 (1mg/ml) in 200ul of complete RPMI at 37oC for 4 hrs.
Growth protocol Pure population of double negative and CD4 T cells were isolated by a step-wise magnetic bead isolation (Miltenyi) using bead specific for non-human primate antigens. Briefly, we stained PBMC with CD4-Microbead to isolate CD4 cells and treated the flow-through with CD8-PE and CD16-PE followed by anti-PE Microbeads for CD8 and CD16 depletion. This CD4-CD8-CD16- flow-through was then positive selected for CD3+ cells with CD3-Biotin and anti-biotin Microbead to obtain, CD3+CD4-CD8- (double negative T cells). Purity was checked after each isolation and further experiments were carried out only with >98% pure populations. Cells were rested overnight prior to functional analyses.
Extracted molecule total RNA
Extraction protocol Cells were collected post-stimulation and immediately lysed in RLT buffer (Qiagen) for RNA extraction. RNA was isolated using RNeasy mini kit (Qiagen) and genomic DNA was removed on-column.
Label Cy5
Label protocol cRNA was synthesized from total RNA and Cy3/Cy5 labeled using the Agilent Low Input Quick Amp Labeling procedure, as per the manufacturer’s instructions.
 
 
Hybridization protocol Labeled cRNA was hybridized to the Rhesus Macaque (V2) Gene Expression 4x44K Microarray (Agilent) in the appropriate combinations, including dye-flip and self versus self controls.
Scan protocol Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
Description 73_FNV_Normal
Data processing Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
After scanning the arrays, data was background subtracted and the probe signal normalized both within the array and between the arrays, using the limma package in Bioconductor.
 
Submission date Sep 11, 2012
Last update date Dec 31, 2012
Contact name Ramsey Saleem
E-mail(s) ramsey.saleem@seattlebiomed.org
Phone 2062567446
Organization name Seattle Biomedical Research
Street address 307 Westlake Ave N
City Seattle
State/province Washington
ZIP/Postal code 98199
Country USA
 
Platform ID GPL16026
Series (1)
GSE40781 CD3/CD28 Stimulated versus unstimulated Double-negative T Cells From Sooty Mangabeys

Data table header descriptions
ID_REF
VALUE normalized log10 ratio Cy5/Cy3

Data table
ID_REF VALUE
1 -1.018707536e-001
2 0.000000000e+000
3 -8.343961595e-001
4 -6.316416121e-001
5 -7.583865835e-001
6 -7.366857084e-001
7 -8.750827816e-001
8 -8.290557750e-001
9 -7.440618336e-001
10 -8.716892536e-001
11 -8.481005850e-001
12 -5.958730457e-002
13 1.462154635e-002
14 -1.843662854e-002
15 -9.716773445e-002
16 -5.207675465e-003
17 -1.443100735e-002
18 -6.293619925e-001
19 -2.540886023e-001
20 -4.864208320e-002

Total number of rows: 45220

Table truncated, full table size 1019 Kbytes.




Supplementary file Size Download File type/resource
GSM1001373_252680610073_201107080936_S01_GE2_107_Sep09_1_1.txt.gz 14.6 Mb (ftp)(http) TXT
Processed data included within Sample table
Processed data provided as supplementary file
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap