GSM6454286: HD 3, PBMC, nTconv, bulk ATAC-seq; Homo sapiens; ATAC-seq
Peripheral blood and tumor tissues for Omni-ATAC-seq were obtained from healthy individuals and NSCLC patients who underwent a surgery for curative resection at the National Cancer Center Hospital East. The PBMCs were isolated by the density gradient centrifugation with Ficoll-Paque (GE Healthcare, Chicago, IL, USA). Tumor tissues were cut into small pieces, and single-cell suspensions were prepared with a gentle MACS Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) as previously described (PMID: 31028147). Briefly, after filtrating the lung homogenates with a cell strainer (30 μm), the cells were subjected to a density gradient centrifugation with Ficoll-Paque to separate the tumor cells from TILs. Each T cell subset [naive Treg cells (CD45RA+CD25lowFoxP3low CD4+), eTreg cells (CD45RA–CD25highFoxP3highCD4+), naive Tconv cells (CD45RA+CD25-CD4+), effector Tconv cells (CD45RA-CD25-CD4+), naive CD8+ T cells (CD45RA+CCR7+CD8+) and effector memory CD8+ T cells (CD45RA-CCR7-CD8+) from healthy individuals' PBMCs; and eTreg cells, eTconv cells and effector memory CD8+ T cells from cancer patients' PMBCs and TILs] was sorted with a BD FACSAria Fusion Cell Sorter (BD Biosciences, San Jose, CA) for subsequent Omni-ATAC-seq analyses. Anti-CD3 (clone: UCTH1), anti-CD4 (clone: RPA-T4) and anti-CD45RA (clone: HI100) mAbs were purchased from BD Biosciences. Anti-CCR7 (clone: G043H7) and anti-CD25 (clone: BC96) mAbs were obtained from BioLegend (San Diego, CA). ATLL cell lines (HuT102, MJ, MT-4, and ATL43T) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal calf serum (Biosera, Orange, CA). Recombinant IL-2 (0.5 nmol/L) was added to the culture medium of ATL-43T due to the IL-2-dependency of the cell line. The Omni-ATAC-seq protocol was performed as described previously with minor modifications (PMID: 28846090). In brief, 5000 to 10000 sorted T cells (for clinical samples), 1000 to 10000 sorted T cells (for murine samples), and 50000 cells (for cell lines) were washed with PBS and then lysed. The cell lysates were resuspended in 50 μl once with TD Buffer containing 2.5 μl of Nextera enzyme (Illumina). The transposition reaction was performed at 37°C for 30 min. The products were then purified with a MinElute polymerase chain reaction (PCR) purification kit (Qiagen, Hilden, Germany), and the fragments for the Omni-ATAC-seq library were amplified for five cycles of PCR. The additional number of PCR cycles was determined for each sample after quantification by real-time PCR. The PCR-amplified products were purified with a MinElute PCR purification kit again.