GSM8432409: primary tumor, validation cohort, YCC-081, 3' scRNA-seq; Homo sapiens; RNA-Seq
Single-cell isolation was performed using a Human Tumor Dissociation Kit (Miltenyi Biotec, USA) following the manufacturer's instructions. Fresh surgical tissues were cut into small pieces (<1 mm3) and placed into a gentleMACS C Tube (Miltenyi Biotec, USA) containing a mixture of Enzyme H, Enzyme R, and Enzyme A in RPMI 1640 medium. The tube was then processed on a gentleMACS Dissociator (Miltenyi Biotec, USA) using the GentleMACS programs h_tumor_01 and h_tumor_02, each followed by a 30-minute incubation at 37°C with rotation. The dissociated tissues were filtered through a 70 μm MACS SmartStrainer (Miltenyi Biotec, USA) to achieve a single-cell suspension. The filtrate was centrifuged at 300 × g for 10 minutes at 4°C. The resultant cell pellet was resuspended in 4 mL of RPMI 1640 medium (Thermo Fisher Scientific, USA), and dead cells were removed using Ficoll-Paque PLUS (GE Healthcare, USA). The cells were subsequently washed and resuspended in PBS supplemented with 0.1% bovine serum albumin (BSA, Invitrogen, USA). Cell viability was assessed using the LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen, USA), and the single-cell suspension was further enriched for viability with a Dead Cell Removal Kit (Miltenyi Biotec, USA). Single-cell suspensions were processed on a Chromium system (10x Genomics, USA). For the discovery cohort consisting of 24 samples, we prepared 5' scRNA-seq libraries using the Chromium Next GEM Single Cell 5′ Library and Gel Bead Kit v1.1 (PN-1000165). For the validation cohort of 18 samples, 3' scRNA-seq libraries were generated using the Chromium Next GEM Single Cell 3ʹ v3.1 Library and Gel Bead Kit v3.1 (PN-1000121). The libraries were sequenced on an Illumina NovaSeq 6000 for the 5' scRNA-seq and an Illumina HiSeq 2000 for the 3' scRNA-seq.