GSM7702830: mcherry_enriched_rep1; Danio rerio; RNA-Seq
NRE-eGFP/HS5-mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf, embryos were anaesthetised with Tricaine (20–30 mg/l) and placed into Danieau's solution. Eyes were dissected from ~100-150 embryos using fine forceps (Dumont #5SF), and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C, then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution, and once with FACSmax (Amsbio). In a final 500 µl FACSmax, the samples were passed through a 35 µm cell strainer to obtain single cell suspension (on ice). Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II (BD) or CytoFLEX SRT (Beckman Coulter) machine. Forward and side scatter sorting was used to select single cells from clumps and debris, and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3' gene expression technology (v3.1).