Ezh2 knockout and gene expression profiling in CD8+ T cells

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. 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.