GSM8412848: gnrh3 knockout, scRNAseq; Danio rerio; RNA-Seq
Five pituitaries were dissected from each gnrh3–/– and WT females at 8 00 h which was 1 hour before lights on. The dissected pituitaries were pooled by genotype in a 1.5 mL Eppendorf tube filled with sterile phosphate-buffered saline (PBS) and left on ice until processing. Pituitary dissociation was performed by following previously described protocols in Hollander-Cohen et al (2021) and Fabian et al (2020) with some modifications. Briefly, the samples were centrifuged at 2,000 × rpm for 5 minutes at 4°C, then PBS was removed followed by a treatment of dissociation solution (0.25% Trypsin; 1 mM EDTA; 0.2% collagenase in PBS; filtered with a 0.22 µm membrane filter) at 28.5°C for 40 minutes, with gently pipetting every 10 minutes using a wide bore tip. After incubation, the treatment was stopped by adding 200 µL 6×Stop solution [6 mM CaCl2; 30% fetal bovine serum (FBS) in PBS] and gently mixed it by pipetting, followed by centrifuging at 2,000 × rpm for 5 minutes at 4°C. The supernatant was gently discarded, then 1 mL Accumax working solution (30% Accumax in PBS) was added, and the cells were gently resuspended. After incubation at room temperature for 5 minutes, the dissociated cells were filtered through 40 µm pore cell strainer. After centrifuging at 2,000 × rpm for 5 minutes at 4°C, the supernatant was removed, and the cell pellets were gently resuspended with 50 µL 0.1% BSA in PBS and kept on ice until processing. cDNA libraries were prepared using 10×Genomics Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 on the 10×Genomics Chromium Controller according to the manufacturer's manual for a target of 4,000 cells per sample.