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Series GSE40741 Query DataSets for GSE40741
Status Public on Dec 20, 2018
Title Ezh2 knockout and gene expression profiling in CD8+ T cells
Organism Mus musculus
Experiment type Expression profiling by array
Summary We used 6 male 4 week old C57BL/6 (B6) mice, three with wild-type (WT) genes, the other 3 with conditional knockout (KO) of both Ezh2 alleles. Knockout was achieved by breading B6 mice with floxed alleles of Ezh2 (Ezh2-fl/fl) to B6 mice expressing Cre recombinase under the control of a CD4 promotor to generate T-cell specific homozygous Ezh2 conditional knockout (CKO) mice. CD8 T-cells were purified using anti-mouse CD8 antibody (Ab)-conjugated magnetic beads from the spleen and lymph nodes of WT and CKO mice. The purity was consistently more that 93% as assessed by flow cytometry analysis. Freshly isolated mature T cells were named naïve CD8 T cells. These naïve CD8 T cells (two million cells / ml) were cultured in media containing 10% fetal bovine serum-IMDM in the presence of plate-coated anti-CD3 Ab (3 mg /ml) + anti-CD28 Ab (3 mg /ml). Three days later, these in vitro stimulated CD8 T cells were collected and named activated CD8 T cells. About 200 ng total RNA was extracted, and amplified using small-sample methods to make biotin-labeled cDNA targets that were hybridized to Affymetrix HT Mouse 430 PM arrays. This resulted in 4 groups of samples: naive or activated cells that were either KO or WT for Ezh2, each group having three biological replicates. The samples are in theory paired, since pairs of samples came from the same mouse, however we did not find many significant mouse to mouse effects, and do not think they need to be modeled. We provide a supplementary Excel workbook that holds the expression data, but it also holds probe-set annotation, and some statistical calculations (and the functions used to compute them), and may be convenient for some users.
As of Dec 20, 2018, we were not expecting to use this data in a publication in the near future, and so released it to the public.
 
Overall design We used 6 male 4 week old C57BL/6 (B6) mice, three with wild-type (WT) genes, the other 3 with conditional knockout (KO) of both Ezh2 alleles. Knockout was achieved by breading B6 mice with floxed alleles of Ezh2 (Ezh2-fl/fl) to B6 mice expressing Cre recombinase under the control of a CD4 promotor to generate T-cell specific homozygous Ezh2 conditional knockout (CKO) mice. CD8 T cells were purified using anti-mouse CD8 antibody (Ab)-conjugated magnetic beads from the spleen and lymph nodes of WT and CKO mice. The purity was consistently more that 93% as assessed by flow cytometry analysis. Freshly isolated mature T cells were named naïve CD8 T cells. These naïve CD8 T cells were cultured in media containing 10% fetal bovine serum-IMDM in the presence of plate-coated anti-CD3 Ab (3 mg /ml) + anti-CD28 Ab (3 mg /ml). Three days later, these in vitro stimulated CD8 T cells were collected and named activated CD8 T cells. About 200 ng total RNA was extracted, and amplified using small-sample methods to make biotin-labeled cDNA targets that were hybridized to Affymetrix HT Mouse 430 PM arrays. This resulted in 4 groups of samples: naive or activated cells that were either KO or WT for Ezh2, each group having three biological replicates.
 
Contributor(s) Zhang Y, Kuick R
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Submission date Sep 10, 2012
Last update date Mar 22, 2019
Contact name Rork Kuick
E-mail(s) rork@umich.edu
Phone 734-936-9241
Organization name University of Michigan
Street address University of Michigan, SPH II, 1415 Washington Heights, Room M2533
City Ann Arbor
State/province MI
ZIP/Postal code 48109-2029
Country USA
 
Platforms (1)
GPL11180 [HT_MG-430_PM] Affymetrix HT MG-430 PM Array Plate
Samples (12)
GSM1000443 Naïve Ezh2-wildtype CD8+ Tcells, mouse #1
GSM1000444 Naïve Ezh2-wildtype CD8+ Tcells, mouse #2
GSM1000445 Naïve Ezh2-wildtype CD8+ Tcells, mouse #3
Relations
BioProject PRJNA174793

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE40741_RAW.tar 17.6 Mb (http)(custom) TAR (of CEL)
GSE40741_series_matrix.txt.gz 2.9 Mb (ftp)(http) TXT
GSE40741_supplement1.xlsx.gz 33.1 Mb (ftp)(http) XLSX
Processed data included within Sample table
Processed data are available on Series record
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