GSM8898463: 20-somite stage, wildtype, rep 1; Danio rerio; RNA-Seq
14- and 20-ss wildtype (TU) and Tg(nkx2.5:ZsYellow) embryos were dissociated to single cells. Briefly, 750 – 1000 embryos were collected and their chorions removed by incubation in 1mg/ml Pronase for 3–4 min followed by washing in 0.5X Danieau Buffer.  Yolks were removed in deyolking buffer followed by embryo dissociation in FACSmax Cell Dissociation solution. After passage through a 40 μm strainer, single cells were resuspended in fresh FACSmax Cell Dissociation solution and passed through a fluorescent activated cell sorter (BD FACSMelodyTM Cell Sorter) to separate the ZsYellow positive fraction. Sorted cells were collected in 0.1% BSA D-PBS. The library prep protocol was based on SMART-Seq protocols with modifications. The workflow of the library preparation can be summarized as follows: Reverse transcription with Maxima H Minus RTase at 50°C for 60 min, followed by enzyme inactivation at 85°C for 3min -> post droplet breaking cleanup with MyOne SILANE beads -> Template switching using  Maxima H Minus RTase (incubate for 30' at 25°C, 90' at 42°C and hold at 4°C; with lid set to 50°C.) -> 1X SPRI purification -> cDNA amplification with PCRI Fwd Primer V2 and PCRI Rev Primer -> 0.6X SPRI purification. The gene expression libraries were made by subjecting the amplified cDNA to Fragmentation, end repair and A-tailing, followed by ligation using NEBNext UltraII FS DNA Library Prep kit for Illumina -> 0.8X SPRI cleanup -> sample indexing by PCR -> double-sided SPRI cleanup.