title: GSM9504660: P16_MetLN_scRNA; Homo sapiens; RNA-Seq
library_construction_protocol: Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient centrifugation using Ficoll-Paque (Cytiva). Tumor and lymph node tissues were minced thoroughly with surgical scissors and transferred to GentleMACs C Tubes containing 3 mg/ml Collagenase IV (Sigma-Aldrich) and 0.1 mg/ml DNase I (Sigma-Aldrich) in RPMI 1640 medium per 0.3g tissue. The tubes were then placed on the GentleMACs Octo Dissociator (Miltenyi Biotec) and incubated at 37°C for 20 min for lymph node samples or 25 min for tumor samples, according to the manufacturer's instructions. After incubation, the samples were quenched by adding 15 ml of RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The cell suspensions were filtered through 70-μm cell strainers (Corning) to remove debris and centrifuged at 300×g for 5 min at 4°C to pellet the cells. When necessary, red blood cell lysis was performed using Red Blood Cell Lysis Solution (Miltenyi Biotec) according to the manufacturer's protocol. Single-cell suspensions with viable cell rates greater than 80% were used for sequencing. The cell suspension was used for 10x Chromium Single cell 5' (5' Kit v2, 1000263) and human TCR/BCR library (TCR 1000252; BCR 1000253) construction as per the manufacturer's instructions. Purified libraries were subsequently sequenced using an Illumina NovaSeq 6000 sequencer with 150-base pair (bp) paired-end reads.