GSM7937496: Sample 11-P; Homo sapiens; RNA-Seq
Tissues were transported in MACS tissue storage solution (Miltenyi Biotec) on ice within 40 min to preserve viability. Samples were rinsed with ice-cold PBS, minced on ice to pieces of less than 1 mm3 and then enzymatically dissociated for 15 min for metastatic liver tumor samples and 10 min for control liver samples at 37°C (with manual shaking every 5 min). Dissociation was carried out in DMEM containing 2 mg/mL collagenase P (Roche) and 0.2 mg/mL DNase I (Roche) as digestion medium for the tumor tissues. Collagenase III (1 mg/mL, Sigma-Aldrich) and hyaluronidase (0.1 mg/mL, Sigma-Aldrich) were additionally introduced to the digestion medium containing metastatic cancer samples to increase cell viability. The digestion suspension was then filtered using a 40 μm nylon cell strainer (Falcon), followed by centrifugation at 300× g and 4°C for 5 min. The cell pellet was resuspended in red blood cell lysis buffer (Sigma-Aldrich) for 5 min at room temperature to remove red blood cells. Samples were centrifuged (250× g, 4°C, 5 min) and resuspended in 200-500 μL (estimated) PBS containing 0.4% BSA (Sigma-Aldrich) and filtered through 40 μm cell strainers. Finally, the concentration and viability of single-cell suspension were checked by a LUNA automated cell counter (Logos Biosystems), and the cells were diluted with PBS containing 0.04% BSA to about 700-1,200 cells/μL The single cells were loaded (aiming at 5,000 cells/sample for each chip position) onto and barcoded with a 10× Chromium Controller (10× Genomics) followed by library construction for scRNA-seq using the Chromium Single Cell 5′ Library and Gel Bead kit (V1.1) from 10× Genomics according to the manufacturer's instructions. The 5' gene expression libraries were sequenced on a NovaSeq 6000 system (Illumina) until sufficient saturation was reached (77.03% on average)