anti-Eomes ChIPseq of control OT-I cells and Eomes-overexpressing OT-I cells

High amount of Eomes might drive T cell exhaustion. In order to understand how Eomes contributes to exhaustion of CD8+ T cell in the TME as a transcription factor, we conducted anti-Eomes ChIPseq analysis of control OT-I cells and Eomes-overexpressing OT-I cells. Eomes was cloned into a retroviral expression vector (RVKM) which also encodes an IRES-hCD2 cassette. This vector was transfected into Pheonix to package retrovirus. The empty vector was used as a control. CD8+ T cells were isolated from spleen and lymph nodes of OT-I mice using Dynabeads Flowcomp mouse CD8 kit (Invitrogen). Then the cells were stimulated with SIINFEKL peptide (OVA257-264) at 2.5 ng/mL in the presence of 10U/mL IL-2 for 24 hr. Retroviral supernatants were harvested, filtered, and supplemented with 6μg/mL polybrene. OT-I T cell cultures were spinduced with the retroviral supernatant for 90 min at 1800 rpm, 32ºC. 48 hr later, hCD2+ cells were sorted prior to re-stimulation. hCD2+ OT-I cells were plated at 40,000 cells/well in 96-well plates and re-stimulated with 2.5ng/mL OVA with 10U/mL IL-2 for 3 days before harvested for ChIPseq analysis. Re-stimulated control or Eomes-overexpressing OT-I cells were harvested, fixed with 1% formaldehyde for 10min at room temperature and stopped by adding glycine to 125mM. ChIP experiment was done following instructions of Active Motif’s ChIP assay kit (53035) with slight modifications, in which 10 cycles of sonication (Bioruptor) were added after enzyme digestion, and dynabeads protein G (Life Technologies) were used for immunoprecipitations. 5μg anti-TBR2/Eomes antibodies (ab23345, abcam) were used for each reaction. The precipitated DNA was sent to BGI genomics for library construction and deep sequencing. To analyze the sequencing data, the sequencing reads were mapped to the Mus musculus genome (version mm10) using Bowtie2 with no more than two mismatches. After PCR duplicates removal by samtools, MACS2 was used for peak calling, compared with each input control with FDR less than 0.001.