GSM8061694: zebrafish dEN dissected tissue; Danio rerio; RNA-Seq
Dissection was performed on 6 individuals of TgBAC(npy:GAL4VP16);Tg(UAS:GFP) fish and 12 dissected tissue pieces from the left and right hemispheres were obtained in total. The dEN and its surrounding regions were carefully dissected with micro-scissors and fine forceps in ice-cold and oxygenized Neurobasal medium (ThermoFisher Scientific 21103049) supplemented with 1x B-27 (ThermoFisher Scientific 17504044) under a fluorescent dissection microscope, as shown in the Figure S5A. The dissected tissue was dissociated with the Papain Dissociation Kit (Worthington; LK003150) with 0.1% 2-mercaptoethanol for 15 minutes with gentle shaking at 28.5 degrees Celsius. Then, the cells were dissociated by gentle trituration 15 times with a glass Pasteur pipet coated with 2% BSA in PBS and spun at 300xg for 5 minutes. The cells were resuspended in papain inhibitor solution (Worthington) and incubated for 10 minutes with gentle shaking at 28.5 degrees Celsius. Then, the cells were further dissociated by gentle trituration 20 times with a glass Pasteur pipet attached with a regular 200-µl tip coated with 2% BSA in PBS. The dissociated cell suspension was then filtered with pluriStrainer Mini 40 µm (pluriSelect) coated with 2% BSA in PBS and spun at 300xg for 5 minutes. The resulting cell suspension was resuspended in 2% BSA in PBS, and then cell debris and dead cells were removed by FACS (FACSAria SORP, BD Biosciences) using Hoechst (to sort out cells from cell debris) and Propidium Iodide (to sort out living cells from dead cells). After FACS sorting, a small fraction of the cell suspension was used to estimate the total number of the cells and their viability using a dead cell stain Trypan Blue (elabscience). The obtained suspension contained 16,000 cells with 85.0% viability. The resulting single-cell suspension was loaded on the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10x Genomics, PN-1000269), and the cDNA library was prepared according to the manufacturer's instructions. The obtained cDNA library underwent Next generation sequencing by illumina Hiseq X (GENEWIZ) with 400,429,716 total reads and 85.9% of sequencing saturation. The obtained sequence was then analyzed by “Cell Ranger count” pipeline provided by 10x Genomics with default options. The reads were aligned to zebrafish reference transcriptome (ENSEMBL Zv11, release 99) and EGFP CDS, which were built by “Cell Ranger mkref” command based on zebrafish reference genome GRCz11 and annotation Ensembl 99. This resulted in 3,381 estimated number of cells with 4890 median unique molecular identifier (UMI) counts per cell.