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Sample GSM404388 Query DataSets for GSM404388
Status Public on May 11, 2012
Title Nstim_2C
Sample type RNA
 
Source name Murine FACS-sorted naïve (CD44lo) CD8 T cells cultured 4hrs ex vivo, stimulated with PMA+ionomycin
Organism Mus musculus
Characteristics strain: C57BL/6N
gender: Female
cell type: CD8+Thy1.2+CD44lo
Treatment protocol FACS-purified naïve (CD44lo) and memory phenotype (CD44hi) CD8+ T cells were cultured for four hours in media alone (Iscove’s DMEM + 8% FBS (HI) + 100U/mL Pennicillin + 100ug/mL Streptomycin + 2mM L-glutamine + 50uM β-mercaptoethanol) or in media with PMA (10ng/mL) and ionomycin (500ng/mL).
Growth protocol n/a
Extracted molecule total RNA
Extraction protocol Following 4hr culture, cells were lysed in TRIzol reagent followed by phenol+chloroform extraction and purification using RNEasy mini columns (QIAGEN).
Label Cy3
Label protocol Approximately 200 ng of total RNA was amplified and labeled with Cy3 using the Low RNA Input Linear Amp Kit PLUS, One-Color (Agilent Technologies, CA). This labeling reaction produces 0.25 – 2.0 µg of Cy3-labeled cRNA (anti-sense), by first Converting mRNA primed with an oligo (d)T-T7 primer into dsDNA with MMLV-RT and then amplifying the sample using T7 RNA Polymerase in the presence of Cy3-CTP.
 
Hybridization protocol After purification, 1.65 μg of cRNA was fragmented and hybridized to the array for 17 hr. at 65°C.
Scan protocol Microarray slides were hybridized overnight, then washed and scanned with Agilent G2565BA Microarray Scanner. Images were analyzed with Feature Extraction 9.5 (Agilent Technologies, CA).
Description Murine FACS-sorted naïve (CD44lo) CD8 T cells cultured 4hrs ex vivo, stimulated with PMA+ionomycin
Data processing Mean foreground intensities were obtained for each spot and imported into the mathematical software package “R”, which was used for all data input, diagnostic plots, normalization and quality checking steps of the analysis process using scripts developed in-house by Peter White specifically for this analysis. In outline, the Cy3 (green) intensities were not background corrected (this has been shown to only introduce noise), and corrected for the scanner offset (40 was subtracted for each intensity). The dataset was filtered to remove positive control elements and any elements that had been flagged as bad. Using the negative controls on the arrays, the background threshold was determined and all values less than this value were set to the threshold value. Finally, the data was normalized using the Quantile Normalization package in “R” (Bolstad et al., 2003).
 
Submission date May 18, 2009
Last update date May 11, 2012
Contact name Joanna DiSpirito
E-mail(s) hshen@mail.med.upenn.edu, rdo2@mail.med.upenn.edu
Organization name University of Pennsylvania
Department Microbiology
Lab Hao Shen, PhD
Street address 3610 Hamilton Walk, University of Pennsylvania
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
 
Platform ID GPL4134
Series (1)
GSE16145 Expression analysis of resting and stimulated naïve and MP CD8 T cells

Data table header descriptions
ID_REF
VALUE normalized signal

Data table
ID_REF VALUE
13 78
14 34
16 561
20 297
21 71
22 101
23 30
24 97
25 1738
26 38
27 280
29 46
34 75
36 2190
37 930
38 11773
39 185
40 462
41 884
42 42

Total number of rows: 33797

Table truncated, full table size 318 Kbytes.




Supplementary file Size Download File type/resource
GSM404388.txt.gz 9.1 Mb (ftp)(http) TXT
Processed data included within Sample table

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