GSM6569752: Tumor-2, NPSCC lymph node (PR); Homo sapiens; RNA-Seq
Tumors were rapidly dissociated after the surgical procedure using the Miltenyi Biotec Human Tumor Dissociation kit (cat# 130-095-929). Each lesion was minced into 1-2mm fragments with surgical scissors in a separate 1.5ml Eppendorf tube containing 932µl DMEM, 42µl of enzyme H, 21µl od enzyme R, and 5µl of enzyme A. Next, tubes were briefly vortexed and placed in a thermomixer for 15 minutes at 37°C, 350rpm. Finally, the dissociated samples were washed through a 50µm filter and counted using a manual hemocytometer to determine cell viability and concentration. Libraries were constructed using the VDJ NextGEM v1.1 10x Genomics Chromium kit according to the manufacturer's instructions. Assessment of libraries quality was performed after cDNA and library generation, measuring the nucleic-acid concentration (Qubit dsDNA High Sensitivity kit; Invitrogen, cat# Q32854) and size distribution (High Sensitivity BioA DNA kit; Agilent, cat# 5067-4626).