GSM5353203: WL02; Homo sapiens; RNA-Seq
Tumours obtained from the surgery were freshly subjected to single-cell dissociation using Human Tumour Dissociation Kit (Miltenyi Biotec) according to the manufacturer's instructions. Briefly, the tumours were washed with plain RPMI (ThermoFisher Scientific) supplemented with Penicillin and Streptomycin (ThermoFisher Scientific), and processed into pieces (1 mm3) using scalpels with the recommended volume of enzyme mix as per instructions. The tumour pieces and enzyme mix were then transferred into gentleMACS™ C tubes (Miltenyi Biotec), and then the tubes were loaded onto the gentleMACS™ Octo Dissociator with Heaters (Miltenyi Biotec) with the program according to the manufacturer's instructions. After incubation, digested tissues were filtered through Falcon® 70-μm strainers (Corning) and washed. Cell suspension was further filtered through 40-μm strainers to remove cell clumps. The viability was assessed by ReadyProbe® Cell Viability Imaging Kit (ThermoFisher Scientific) to ensure the viability was greater than 90%. Single-cell RNA sequencing was performed using Chromium Single Cell 3' Reagent Kits v3 (10x Genomics) according to the manufacturer's protocol. WL02 was aimed with a targeted capture of 2,000 cells due to the limited tissue obtained from the surgery, while WL03 with a targeted capture of 9,000 cells. Gel Beads-in-emulsion was generated using Chromium Single Cell B Chip Kit. Libraries were sequenced using the Illumina NovaSeq 6000 and S2 flow cells (100 cycle kit) with a read one length of 28 cycles and a read two length of 94 cycles.