cell type: CD8+ T cells cd8+ t cell subset: TSCM donor: 3
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#3 TSCM Donor#3 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
