GSM7080445: P30, negative; Homo sapiens; RNA-Seq
We collected fresh tumor tissues which was mechanically and enzymatically dissociated using a gentleMACS dissociator (Miltenyi Biotec, Gladbach Bergisch, Germany, Cat#130-093-235) and the Human Tumor Dissociation Kit (Miltenyi Biotec, Cat#130-095-929) following the manufacturer's instructions. After incubation for 1 h at 37℃, the resuspended samples were filtered through a 70-µm MACS SmartStrainer (Miltenyi Biotec, Cat# 130-098-462) into RPMI-1640 medium (Corning, Inc. Corning, NY, USA) supplemented with 10% fetal bovine serum (Biowest, Riverside, MO, USA) and centrifuged at 300 ×g for 10 min. Libraries were prepared using the Chromium controller according to the 10x Chromium Next GEM Single Cell 5-v2_CellSurfaceProtein_UserGuide (CG000330). Briefly, the cell suspensions were diluted in nuclease-free water to achieve a targeted cell count of 10,000. Cell suspension was mixed with master mix and loaded with Single Cell 5′ Gel Beads and Partitioning Oil into a Next GEM Chip K. RNA transcripts from single cells were uniquely barcoded and reverse-transcribed within droplets. cDNA molecules were pooled and enriched with PCR. The amplified cDNA are separated by size selection for 5ʹ Gene Expression libraries, V(D)J Enriched Libraries and Cell Surface Protein libraries