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Sample GSM572426 Query DataSets for GSM572426
Status Public on Jul 13, 2011
Title Stem cell memory (TSCM) cells from Donor#2 [gene-level]
Sample type RNA
Source name CD8+ T cells
Organism Homo sapiens
Characteristics cell type: CD8+ T cells
cd8+ t cell subset: TSCM
donor: 2
Treatment protocol N/A
Growth protocol Peripheral blood mononuclear cells (PBMC) were isolated from lymphocyte-enriched apheresis blood (provided by the NIH Blood Bank) according to standard techniques and used immediately for cell sorting. CD8+ T cells were enriched from PBMC by using the EasySep CD8+ T cell enrichment kit (Stem Cell Technologies), stained with different combinations of fluorescent-labeled monoclonal antibodies and sorted with a FACSAria (BD Biosciences) in different live (Live/Dead-; Invitrogen) CD8+ T cell subsets, as follows: TN (CD45RO-, CD45RA+, CCR7+, CD27+, CD62L+, CD11a dull, CD95-), TSCM (CD45RO-, CD45RA+, CCR7+, CD27+, CD62L+, CD11a dull, CD95+), TCM (CD45RO+, CD62L+, CCR7+) and TEM (CD45RO+, CD62L-, CCR7-), generating 12 samples total.
Extracted molecule total RNA
Extraction protocol RNEasy Micro kit (Qiagen), following manufatcturer's instructions
Label biotin
Label protocol WT expression kit (Ambion), WT Terminal Labeling Kit (Affymetrix), according to the manufatcturer's instructions
Hybridization protocol Samples were hybridized to WT Human Gene 1.0 ST arrays using Affymetrix hybridization kit materials, according to the manufacturrer's instructions. Sample cocktails were heated at 99°C for 5 minutes, then 45°C for 5 minutes, centrifuged at max speed for 1 minute. Eighty ul of hyb solution was transferred to each array, and hybridized 17 hours at 45°C at 60rpm. Fluidics washing protocol used was FS450_0007.
Scan protocol Arrays were scanned on a GeneChip Scanner 3000 7G (Affymetrix).
Description FACS-sorted CD8+ T cell subset derived from healthy blood donor
Donor#2 TSCM
Donor#2 Tcd95_(HuGene-1_0-st-v1).CEL
Data processing In all processing steps, samples were processed at the same time as a single batch. Raw data from the generated .CEL files were imported into Partek Genomincs Suite using the RMA method.
For exon-level analysis, exons differentially expressed between groups (i.e. genes subject of alternative splicing) were identified using Partek's Alternative Splicing ANOVA (p<0.05) supported by the Benjamini-Hochberg correction method for multiple comparisons allowing for a false discovery rate of <0.05.
For gene-level analyses, probeset level data were merged into gene-level datasets based on median probeset signal level.
Differentially expressed genes (DEGs) were identified by One-Way Repeated Measures ANOVA (p<0.01) corrected by Benjamini-Hochberg's False Discovery Rate method (p<0.05), resulting in a gene list of 900 DEGs. For pair wise comparisons between all possible sample pairs (e.g. TN vs. TSCM, etc), this gene list was further filtered for between-group alpha levels of p<0.01 and a fold change criterion of >2.0. By additional filtration of the 900 significant genes, a separate list of DEGs, displaying gradually changing expression levels along with the process of memory cell differentiation, i.e. in the TN -> TSCM -> TCM -> TEM direction, direction, was also identified. Finally, a limited list of common DEGs significant when comparing T naive CD95+ cells with all three other T cell populations individually was also described using slightly modified selection criteria; One-Way Repeated Measures ANOVA (p<0.05) corrected by Benjamini-Hochberg's False Discovery Rate method (p<0.05).
Exon-level analysis_probe group file: HuGene-1_0-st-v1.r4.pgf
Transcript-level analysis_meta-probeset file: HuGene-1_0-st-v1.r4.mps
Submission date Jul 29, 2010
Last update date Jul 13, 2011
Contact name Zoltan Pos
Phone +(36)12102930-56435
Organization name Semmelweis University
Department Dept. of Genetics, Cell- and Immunobiology
Street address 4 Nagyvarad ter
City Budapest
ZIP/Postal code H-1089
Country Hungary
Platform ID GPL6244
Series (1)
GSE23321 Expression data from human naïve (TN), stem cell memory (TSCM), central memory (TCM) and effector memory (TEM) CD8+ T cells
Affiliated with GSM572414

Data table header descriptions
VALUE Log 2-transformed RMA signal intensities from Partek GS

Data table
8157281 5.82199
7997332 7.93272
8072798 9.98417
7972808 6.84816
8157283 8.7541
7947375 3.7479
7972810 8.69641
8097335 4.24468
8047377 3.30863
7997336 5.36486
8047379 5.24364
8047381 9.03942
8022856 4.95918
8157300 4.96441
8072817 7.20619
7997346 4.90788
8107348 5.66985
8157308 6.01182
8107350 9.26369
8072825 8.09934

Total number of rows: 28869

Table truncated, full table size 447 Kbytes.

Supplementary data files not provided
Processed data included within Sample table

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