title: GSM4838132: Visium-B; Danio rerio; RNA-Seq
library_construction_protocol: For spatial transcriptomics using the Visium platform from 10X genomics: Adult tumor-bearing fish were euthanized on ice and washed in 1X PBS. After dissection of the head and tail, the remaining tissue was equilibrated in cold OCT for 2 minutes, before transfer to a tissue mold filled with fresh OCT for snap-freezing in liquid nitrogen-chilled isopentane. Tissue blocks were stored at -80°C. For cryosectioning, both the tissue block and the ST slide were equilibrated inside the cryostat for 15-30 minutes at 16°C before sectioning. Transverse sections through the entire fish were cut at a thickness of 10 µm and immediately placed on the ST slide (Visium Spatial Gene Expression Slides, 10X Genomics). ST slides containing sections were stored at -80°C for a maximum of one week before use. For single-cell RNA-seq using the 10X genomics 3' GEX (v3) platform: Adult tumor-bearing fish were dissected to obtain only the tumor and surrounding tissues (i.e head and tail were removed). Tissue was minced with a fresh scalpel and incubated in 0.16 mg/mL liberase (Sigma-Aldrich #5401020001) in 0.9X PBS for 15 minutes at RT. Tissue was then further dissociated by repeated pipetting with a wide-bore P1000, followed by incubation for an additional 15 minutes at RT. After adding 500 uL FBS to stop the dissociation reaction, samples were filtered through a 70 µm filter and centrifuged at 500g for 5 minutes at RT. The resulting pellet was resuspended in DMEM supplemented with 2% FBS, and cells were sorted at room temperature to remove debris and doublets, using a BD FACSAria III cell sorter (BD Biosciences). Equal numbers of GFP+ (tumor) and GFP- (macroenvironment) cells were collected.  For spatial transcriptomics using the Visium platform from 10X genomics: Samples were processed according to the Visium Spatial Gene Expression User Guide (10X Genomics) and all reagents were from the Visium Spatial Gene Expression Kit (10X Genomics). Briefly, sections were fixed in chilled methanol for 30 min at -20°C, stained with hematoxylin and eosin, and mounted in 85% glycerol for imaging. Imaging was performed on a Leica SCN400 F whole-slide scanner at 40X magnification. After imaging, sections were permeabilized at 37°C for 45 minutes. After permeabilization, the on-slide reverse transcription (RT) reaction was performed at 53°C for 2 hours. Permeabilization time and RT reaction length were determined using the Visium Spatial Tissue Optimization Kit (10X Genomics). Second strand synthesis was subsequently performed on-slide for 15 minutes at 65°C. All on-slide reactions were performed in a thermocycler with a metal slide adaptor plate. Following second strand synthesis, samples were transferred to tubes for cDNA amplification and cleanup. Library quality was assayed using a Bioanalyzer High Sensitivity chip (Agilent). For single-cell RNA-seq using the 10X genomics 3' GEX (v3) platform: Equal numbers of sorted GFP+ (tumor) and GFP- (macroenvironment) cells were centrifuged at 300g for 5 minutes at RT, and resuspended in DMEM + 10% FBS. Droplet-based scRNA-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 10,000 cells from two fish were split encapsulated in a single v3 reaction. GEM generation and library preparation were performed according to manufacturer's instructions.