GSM5380072: Nuc-seq; Danio rerio; RNA-Seq
Adult tumor-bearing fish were dissected to obtain only the tumor and surrounding tissues (i.e head and tail were removed). The tissue was then ground in a Dounce homogenizer on ice in Nuclei EZ Prep Lysis Buffer (Sigma-Aldrich #NUC101). The nuclear suspension was then spun down at 4°C (500g, 5 min). After resuspending the pellet in 1 mL wash buffer (250 mM sucrose, 50 mM citric acid, 1% BSA, 20 mM DTT, 0.2U/µL RNAse inhibitor), the sample was again spun at 4°C for 5 min at 500g. The pellet was resuspended in 1 mL wash buffer and subsequently sorted at 4°C to isolate individual nuclei, using a BD FACSAria III cell sorter (BD Biosciences). Approximately equal numbers of GFP+ (tumor) and GFP- (microenvironment) nuclei were collected. Equal numbers of sorted GFP+ (tumor) and GFP- (microenvironment) nuclei were centrifuged at 600g for 5 minutes at 4°C. Droplet-based snRNA-seq was performed using the Chromium Single Cell 3' Library & Gel Bead Kit v3 (10X Genomics) and Chromium Single Cell 3' Chip G (10X Genomics). Approximately 12,000 nuclei were encapsulated in a single v3 reaction. GEM generation and library preparation were performed according to manufacturer's instructions. 