GSM8464445: cranial NCCs, YM; Danio rerio; RNA-Seq
Cranial neural crest cells were isolated from zebrafish embryos using a fli1a:EGFP and sox10:mRFP double labeling technique as described in (Barske et al., 2016; Askary et al., 2017; Mitchell et al., 2021) with some modifications. In brief, 140 double positive embryos were dechorionated at 14 hpf with 2 mg/ml Pronase (Roche, cat. 10165921001), then 70 embryos were treated with 100 uM YM-254890 (Adipogen, cat. AG-CN2-0509-M001) or DMSO (control) from 16-36 hpf. At 36 hpf, 10 embryos from each condition were set aside for skeletal preparations at 6 dpf. For the remaining embryos, the heads were severed, pooled, and deyolked by gentle pipetting in Ca2+-free Ringer's solution (116 mM NaCl, 2.9 mM KCl, 5 mM HEPES, pH 7.0). The embryos were then dissociated into single cells with mechanical agitation and enzymatic digestion for 15 minutes at 4˚C with cold-activated protease from Bacillus licheniformis (Sigma Aldrich, cat. P5380) in dissociation buffer (10 mg/ml Bacillus licheniformis protease, 125 U/ml DNAse, 2.5 mM EDTA, PBS), followed by enzyme neutralization with stop solution (30% fetal bovine serum, 0.8 mM CaCl2, in PBS). Cells were then centrifuged (400xg), resuspended in cell suspension buffer [1% fetal bovine serum, 0.8 mM CaCl2, Leibovitz's L-15 Medium (Gibco, cat. 21083-027)], filtered through a 70 uM strainer (PluriSelect, cat. 43-10040-40), centrifuged (400xg) again and resuspended in sorting buffer (1% fetal bovine serum, 1 mM EDTA, 25 mM HEPES). EGFP and mRFP double positive cells were then purified with fluorescence activated cell sorting using MoFlo XDP100 (Beckman Coulter). A total of 72,000 and 86,000 double positive cells were purified for DMSO and YM-treated samples, respectively. Cells were once again centrifuged (400xg) and resuspended in cell suspension buffer. After confirming cell viability with trypan blue (Gibco, cat. 15250-061), 8000 cells for each condition were submitted to the Genomics and Microarray Core (CU AMC) for single cell RNA-sequencing. Library was prepared according to manufacturer's protocol (3' NextGEM V3.1, 10X Genomics)