GSM3067193: Zebrafish Embryos, 14hpf; Danio rerio; RNA-Seq
Zebrafish embryos were incubated to the indicated times post-fertilization and chorions were removed by incubating in 1mg/mL Pronase (Sigma P5147-1G) for 3-4 min followed by washing in 0.3X Danieau Buffer. [10X Danieau Buffer = 174 mM NaCl, 2.1 mM KCl,1.2 mM MgSO4, 1.8 mM Ca(NO3)2, 15 mM HEPES,  pH 7.6].  Dissociation of embryonic tissues was performed similarly as previously described (Manoli & Driever, Cold Spring Harbor Protocols 2012) with the following specifications. Wild-type and CRISPR-targeted samples were each prepared from 20-100 embryos. Embryo tissues were triturated to homogeneity in 1-5mL FACSmax cell dissociation solution (Genlantis T200100) and incubated for 4-5 minutes at room temperature.  Cells were then filtered through a 40μm cell strainer mesh (Fisher 352340), and centrifuged in a swinging bucket rotor at 310g for 5 minutes.  Cell pellets were resuspended in 1X DPBS (no Ca/Mg, Life Technologies 14190-144) containing 1% BSA (Sigma A3311-100G), and subjected to 2-3 additional rounds of centrifugation and resuspension.  After washing, cells were resuspended in 0.5% BSA / DPBS containing 18% optiprep density medium (Sigma D1556-250ML).  Cell density was quantified manually using INCYTO™ C-Chip™ Disposable Hemacytometers (Fisher 22-600-100), and adjusted to ~100,000 cells per mL.  For single-embryo dissociations, all FACSmax and wash volumes were reduced to a volume 0.5 mL and were carried out in 0.5mL LoBind microcentrifuge tubes (Eppendorf 022431005) that had been pre-coated with 10% BSA/DPBS for 15 minutes at room temperature.  Single-cell transcriptomes were then barcoded using the inDrops platform as previously described (Zilionis et al., Nature Protocols 2017). Following the within-droplet reverse transcription step, emulsions were split into batches of approximately 1,000-2,000 cells, frozen at -80C, and subsequently processed as individual RNA-seq libraries. Single-cell RNA-Seq libraries were prepared using a protocol customized for the inDrops platform (see Zilionis et al., Nature Protocols 2017)