GSM7908445: patient 4, AXLN2 LEC, non-metastatic; Homo sapiens; RNA-Seq
Axillary lymph nodes (AXLNs) were collected from breast cancer patients undergoing mastectomy and AXLN removal. LNs were cut into small pieces, and digested with RPMI containing 0.2 mg/ml collagenase P, 0.8 mg/ml dispase, and 0.1 mg/ml DNase. To confirm the presence of cancer cells in the LNs, a portion of single-cell suspension was stained with AF488-anti-pancytokeratin antibody. Pan-cytokeratin-positive or -negative LNs were considered as metastatic or distant LN, respectively. CD45+ cells were depleted using Human CD45 depletion kit, and the single-cell suspension was incubated with PE-anti-PDPN, AF488-anti-CD45, and APC-anti-CD31. Live CD45- PDPN+ CD31+ LECs were sorted and processed following 10X Genomics guidelines. scRNA-seq libraries were prepared to according to the manufacturer's instructions using Chromium Single Cell 3' Library and Gel Bead Kit v2 and v3 (10x Genomics, 120237) along with Chromium Single Cell A Chip Kit.