GSM9282541: Tg(gnrh3:EGFP) zebrafish, 7 dpf, GnRH3 neuronal nuclei; Danio rerio; RNA-Seq
250 live larvae of Tg(gnrh3:EGFP) were euthanized by chilling on ice for 20 minutes, followed by de-yolking by vigorous pipetting with 200 µl tip using 3 mL swelling buffer (250mM Sucrose, 10mM Tris-HCL (pH 7.9), 10mM MgCl2 and 1mM EGTA). The mixture was passed through a 40 uM tube top into 50 ml falcon tube. The retained embryos were transferred to a tube containing 3.5 mL of cell lysis buffer (Nuclei EZ Lysis Buffer, Sigma) and incubated on ice for 30-40 mins, during which the solution was passed through 3 mL syringe equipped with 22-gauge needle every 5 mins. The mixture was then centrifuged at 500g at 4oC for 5 min and the supernatant was collected twice and pooled supernatant was filtered through 20 µm filter into 15 mL falcon tube. After centrifugation at 500g for 5 min at 4°C, supernatant was removed and then the pellet containing the nuclei was resuspended in 1 mL PBS+1% BSA containing 200 units/mL protector RNase inhibitor (Millipore Sigma, MD, USA). 50 ul 7-AAD stain was added, and the nuclei were then subjected to fluorescence-activated cell sorting (FACS) to enrich GnRH3 nuclei based on EGFP labeling using BD FACS Aria II cell sorters (Nozzle 100 μm) Single-nuclei RNA sequencing (snRNA-Seq) cDNA library was prepared from 10,000 nuclei using Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10×Genomics, Pleasanton, CA, USA) on the 10×Genomics Chromium Controller according to the manufacturer's manual. cDNAs were barcoded followed by the library sequenced using Illumina NovaSeq6000 platform with 100-bp paired-end reads at a depth of 77,538 mean reads per nucleus.