GSM2442543: P14d8_r1; Mus musculus; ATAC-seq
Cell suspensions were prepared from tumors after enzyme digestion.  For sorting of adoptively transferred OT-I and P14 cells, CD8+ T cells were positively selected from tumors using the Dynabeads FlowComp Mouse CD8 selection kit (Thermo Fisher). To ensure adequate cell yield, samples from multiple mice belonging to the same group were pooled prior to enrichment. Enriched CD8+ T cells were then stained with anti-CD8a (53-6.7), anti-CD45.1 (A20), anti-Thy1.1 (OX-7), anti-PD-1 (29F.1A12), anti-Tim-3 (RMT3-23) and Fixable Viability Dye eFluor780. Tumor infiltrating lymphocytes were isolated as live CD8+CD45.1+ (OT-I) or CD8+Thy1.1+ (P14) on BD FACSARIA III or FUSION sorters. ATAC-seq was performed according to Buenrostro et al. (Nature Methods 2013) with minor modifications: 30,000 to 50,000 sorted CD8+ T cells cells were lysed to extract nuclei. Nuclei were resuspended in 50 ul 1xTD Buffer containing 2.5 μl transposase (Nextera, Illumina). The transposase reaction was conducted for 30 minutes at 37°C, with mild shaking. Library amplification and barcoding were performed with the library amplification kit (KAPA Biosystems) using Index Primers, designed according to Buenrostro et al. (Nature Methods 2013) at a final concentration of 500 nM. PCR reaction was conducted for 10-11 cycles. Library purification was performed with the MinElute PCR Purification Kit (Qiagen) and library size distribution was assessed using the Bioanalyzer High Sensitivity DNA Kit (Agilent). ATAC-seq libraries were quantified prior to pooling and sequencing using the real-time library quantification kit (KAPA Biosystems).
GEO Accession
GSM2442543