GSM7729462: Male, smoker, ATAC, subject1; Homo sapiens; ATAC-seq
The tissues were collected at a ~2 cm3 size and put in MACS Tissue Storage Solution (Miltenyl biotec cat.130-100-008) within 30 mins of surgical dissection. Samples were kept at 4°C and processed within 3 hours of dissection. Tissues were chopped into 2 mm diameter pieces within a dissociation tube to reduce the cell loss. Multi Tissue Dissociation Kit 1 and gentleMACS Octo Dissociator (Miltenyi Biotec) were used for dissociation of the tissue into single-cell suspension with a reduced amount of enzyme R (25% of the regular amount). Red blood cells were removed using Red Blood Cell Lysis Solution (Miltenyl biotec cat.130-094-183). Single-cell suspension of samples was frozen using 90% FBS and 10% DMSO and stored in liquid nitrogen until further processing except for the time of transfer in dry ice. The dissociated single-cell suspensions were thawed and filtered using a 70 μm cell strainer (Miltenyi Biotec) to remove debris. The filtered cells were labelled with cell viability marker (DAPI) and antibody markers of EPCAM, CD31, and CD45. Live single-cells (DAPI-negative) were sorted into lysis plates based on three gates: EPCAM+CD45- (designated “epithelial”), EPCAM-CD45+ (designated “immune”), and EPCAM-CD45- (designed “endothelial or stromal”). To enrich epithelial cells, which are considered to have key roles in lung cancer etiology, we collected all “epithelial” cells from EPCAM+CD45- gates and balanced the ratios to 6:3:1 (“epithelial”: “immune”: “endothelial or stromal”). Nuclei isolation was performed based on “Low Cell Input Nuclei Isolation” protocol (CG000365-Rev C, 10X Genomics) with a modification of the 1X lysis buffer treatmenet time (3-second duration). Isolated nuclei were used for single-cell capture and sequencing library preparation using the Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Kits following the manufacturer's guidelines (CG000338- Rev E, 10X Genomics, USA).