CHIP-seq of healthy donor-derived CD8+ T cells in flask and 3D conditions

The healthy donor-derived CD8+ T cells were incubated in anti-CD3/CD28 coated plates for 36-hour activation. Cells were then collected by incubating flask and 3D fibrin gel for 3 days. ChIP was performed by using an iDeal ChIP-seq kit for Histones (Diagenode) according to the manufacturer’s protocol. In brief, 1×107cells were crosslined with 1% formaldehyde for 8 min at room temperature. After stopping the fixation with 0.125M glycine for 5 min at room temperature, cells were washed, lysed and sheared by sonication using Bioruptor (Diagenode) for 20-30 cycles (30s “ON”, 30s“OFF”) at high power setting. The sheared chromatin fragments were immunoprecipitated with protein A-coated magnetic beads and anti-P73; anti-RUNX2; anti-TEAD or anti-IgG (Cell Signaling, Cat.: 3900S; 1:50) antibody. After elution and decross-linking, the enriched DNA fragments were purified by IPure beads v2. According to manufacturer’s protocol, Chip-seq library construction using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme). The library was sequenced with an Illumina NovaSeq 6000 (Illumina).The quality of NSG data was assessed by FastQC. Chip-seq Data were aligned by Bowtie2 to the human reference hg19. For Chipseq, the R package ChIPseeker was used for ChIP peak annotation and comparison.