Genome binding/occupancy profiling by high throughput sequencing
Summary
Here, we present genome-wide comparisons of chromatin accessibility from endogenous CD8+ T cells and transferred OT-1 CD8+ T cells in young vs. aged B16-OVA tumors using ATAC-seq. This ATAC-seq dataset includes three different intratumoral CD8+ T cell subsets, such as TProg: Slamf6+Tim-3-, TTerm: Slamf6-Tim-3+, and TTAD: Slamf6-Tim-3-, as well as naive CD8+ T cells from spleens.
Overall design
Live tumor-infiltrating CD8+ T cells or OT-1 CD8+ T cells from tumors on day 15 after tumor inoculation were sorted and pooled into 2 or 3 independent biological replicates, which ranged from 5,000 to 50,000 cells per genotype/subset combination (TProg: Slamf6+Tim-3-, TTerm: Slamf6-Tim-3+, and TTAD: Slamf6-Tim-3-). Pelleted cells were incubated in 5–50 μl of reaction mix containing 2×TD, Tn5 enzyme, 2% digitonin in nuclease-free water at 37 °C for 30 min with agitation at 1000 RPM. MinElute Reaction Cleanup kit (Qiagen) was used for DNA purification. Following PCR, bead cleanup was done using AMPure XP beads (Beckman Coulter/Agencourt), library quality was verified using Tapestation analysis, and library quantification was confirmed by KAPA Library Quantification Kit Illumina® Platforms (Roche). Sequencing was performed on NextSeq500 or NextSeq 1000 sequencer using paired-end 50 bp reads.
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