GSM8327216: Perturbation experiment 2, 24 hpf biological replicate 1, technical replicate 1 to 4; Danio rerio; OTHER
Zebrafish embryos were dechorionated using 1mg/mL Pronase (Sigma P5147-1G) for 5-7 minutes followed by washing in egg water. Embryos were dissociated as previously described in Wagner et. al. Science 2018. Briefly, embryos were dissociated in 0.5mL LoBind microcentrifuge tubes that had been precoated with 10% w/v BSA for about 15 minutes at room temperature. They were homogenized in 1XDPBS/1% w/v BSA for 6 to 10 hpf embryos (early time points) and in 500 µL FACSmax cell dissociation solution (Genlantis T200100) for 14 to 24 hpf embryos (late time points). Cells were then filtered through a 40µm cell strainer mesh (Fisher 352340) and centrifuged in a swinging bucket rotor at 200g for 1 minute (early time points) or 300g for 5 minutes (late time points). Cells were then washed in 1XDPBS/1%BSA using the same centrifuge settings. They were then resuspended in 1XDPBS/0.5%BSA/18% optiprep density medium (Sigma D1556-250ML). Cell concentration was manually quantified using hemocytometer and adjusted to a final concentration of ~100,000 cells/mL before encapsulation. Library construction as detailed in Zilionis, R. et al. 2016 Nature Protocols. Single-cell barcoding and sequencing was done using droplet microfluidics also described in the same paper.