GSM9010026: 444_NeCh_HuGFP; Danio rerio; RNA-Seq
Embryos were dechorionated and placed into 0.5x Danieau's solution with tricaine (MS-222) anaesthetic. Embryo cranial regions were dissected using a scalpel blade and collected into 0.5x Danieau's solution on ice. The samples were washed twice using 0.5x Danieau's solution and once with FACSmax (Amsbio) after centrifugation at 300 x g for 1 min at 4°C. Finally, samples were resuspended in 500 μL FACSmax and passed through a 35-μm cell strainer to obtain single-cells. Cells were sorted based on fluorescence using the CytoFLEX SRT (Beckman Coulter). Up to 40,000 sorted cells per sample were processed for scRNA-seq and Illumina library preparation using the 10x Genomics Chromium single-cell 3′ gene expression technology (v3.1) by the IGC FACS facility. Sequencing of the resulting libraries was carried out on the NextSeq 2000 platform (Illumina Inc.) using the NextSeq 1000/2000 P3 Reagents (100 cycles) v3 Kit, aiming for ∼50,000 reads per cell.