GSM6448799: NSCLC 13, normal lung, Treg and CD8+ T, scRNA-seq; Homo sapiens; RNA-Seq
Peripheral blood and tumor tissues for scRNA-seq were obtained from healthy individuals and NSCLC patients who underwent a surgery for curative resection at the National Cancer Center Hospital East. Each Treg cell subset [Naive Treg cells (CD45RA+CD25lowFoxP3low CD4+) and eTreg cells (CD45RA–CD25highFoxP3highCD4+)] was sorted with a BD FACSAria Fusion Cell Sorter (BD Biosciences, San Jose, CA) for subsequent scRNA- seq analyses. Naive Treg cells and eTreg cells from healthy individuals' PBMCs, normal lung tissues, and cancer patients' TILs were purified for scRNA-seq analyses using Ficoll-Paque or Tumor Tissue Dissociation Reagent (BD Biosciences), which enzymatically dissociate tissues as previously described (PMID: 35852944  ).  Normal lung tissues were resected from the same lung lobe as the lung tumor, but from the area farthest from the tumor (at least >5 cm from the tumor). Because of the limited number of Treg cells in the normal lung tissues, both Treg cells (CD45RA-CD25highCD4+ T cells) and CD8+ T cells (CD45RA-CCR7-CD8+) in normal lung tissues were sorted, and then CD8+ T cells were bioinformatically removed from the downstream analyses of scRNA-seq and scATAC-seq. 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). Fixable Viability Dye eFluor 780 was purchased from Thermo Fisher Scientific (Waltham, MA). scRNA-seq libraries were constructed with Chromium Next GEM Single Cell 3′Reagent Kits v3.1 (10x Genomics, Pleasanton, CA) according to the manufacturer's instruction. Cell suspensions of each sample were processed in the Chromium Controller (10x Genomics) to generate single-cell gel bead-in-emulsions (GEMs) for sample and cell barcoding.