GSM8816425: Fli1b_mutant; Danio rerio; RNA-Seq
fli1b-/- and wild-type embryos were obtained by the corresponding in-crosses of sibling parents that were either homozygous mutant (fli1b-/-) or wild-type (fli1b+/+). Approximately 40 embryos from each sample were manually dechorionated at approximately 23-24 hpf, then transferred into 1.5mL tubes. Whole embryos were then dissociated into a single-cell suspension by cold protease tissue dissociation protocol (Potter and Potter, 2019). All steps were performed on ice. Embryo water was removed and deyolking buffer was added (55mM NaCl, 1.8mM KCl, 1.25mM NaHCO3, in 1XPBS). Embryos were pipetted up and down until yolk was dissolved, then centrifugated at 300G for 1 min. Supernatant was aspirated and embryos were resuspended in 0.5X Danieau Buffer. This step was repeated and after the supernatant was aspirated, Bacillus lichenformis enzyme mix (1x FBS in PBS, 0.5mM of EDTA, 125 U/mL of DNase 1, 10mg/mL of Bacillus lichenformis) was prepared and added to embryos.  Embryos were incubated on ice for 20 mins and titrated 15 times every 2 min. Cell dissociation was monitored under microscope and once completed, centrifugation was performed at 1,200G for 5 min. Supernatant was discarded and pellet was resuspended in 10% FBS in PBS. Cells were then passed through 20-µm strainer, then washed with 500 ul of 10% FBS. Cells were then pelleted again at 1,200G for 5 min, washed once with 1ml of 10% FBS, and centrifuged at 1,200G for 5 min. Cells were resuspended in 250 ul of 10% FBS, then counted using a hemocytometer. A suspension of approximately 16,000 cells (for each of genotype) was loaded onto the 10x Genomics Single Cell 3' chip at the USF Genomics Core. Library was prepared according to the manufacturer's instructions (single cell 3' v3.1 protocol).