GSM8279213: 02-02T; Homo sapiens; RNA-Seq
Within 4-6 h of tissue acquisition, the specimen was placed in ice-cold RPMI 1640 medium (Gibco, Cat. No. 11875101). Tissue samples were washed in ice-cold phosphate-buffered saline (PBS; Corning, Cat. No. 21- 040-CM) and grossly assessed for adequacy (solid tissue component, free of large hemorrhagic or necrotic areas). Next, the tissue was manually minced with a scalpel blade and filtered through a 40 μm mesh filter (Falcon; Cat. No. 352340) to remove any excess debris or blood. The tissue was then transferred to a sterile gentleMACS C tube (Miltenyi Biotec, Cat. No. 130096334), resuspended in 3-5 mL of Miltenyi Tumor Dissociation Kit buffer (Cat. No. 130-095-929), and mechanically minced to submillimeter particles. The suspension was then digested utilizing the gentleMACS Octo Dissociator (Miltenyi Biotec, Cat. No. 130-095-937) for 25-30 min using the “37C_h_TDK_3'' program for tumor tissue. Dissociated cells were then quenched with ice-cold RPMI 1640 containing 10% fetal bovine serum (FBS; Gibco, Cat. No. 10082147) and filtered through a 40 μm mesh filter. Red blood cells were removed by treatment with 3-5 mL of ACK lysis buffer (Gibco, Cat. No. A1049201) and a subsequent washing step in RPMI 1640 with 10% FBS. Final cell counts were established with 0.4% trypan blue solution and a Countess II Automated Cell Counter (Applied Biosystems, Cat. No. A27977). The final concentration was titrated to 300-500 viable cells/μL for further analysis. Chromium Single Cell 3′ Reagent Kits (v3)—the Single Cell 3′ Library & Gel Bead Kit v3 (PN-1000075) [for patients with prefix 02-] or the 5' kit v2 (PN-1000263) [for patients with prefix 01-], the Chromium Next GEM Chip G Single Cell Kit (PN-100120), and the i7 Multiplex Kit (PN-120262) (10x Genomics)