GSM4859953: GFP_neg_48h_rep1; Danio rerio; RNA-Seq
To isolate heart, embryos were anesthetized with Tricaine (0.16 mg/ml in egg water) and large scale extraction was performed according to the previously published protocol with minor adjustments (Lombardo et al. 2015). GFP-expressing hearts were manually separated from remaining tissues under fluorescence stereomicroscope and collected into 0,5 ml of EDM (L-15/10% FBS). Pool of 300-500 hearts was dissociated with Trypsin-EDTA solution (0.05%) as previously described (Winata et al., 2013). FACS Aria II cytometer (BD Biosciences, USA) was used to enrich GFP positive (fluorescent) and GFP negative (nonfluorescent) heart fractions. Gates for cell sorting were calibrated against dissociated hearts extracted from wild type zebrafish embryos at respective developmental stages (48 or 72 hpf). Cells were sorted directly to 500 µl TRIzol™ LS Reagent (Thermo Fisher Scientific, USA) followed by RNA purification and DNase I treatment by means of Direct-zol™ kit (Zymo Research, USA) according to the manufacturer's protocol. To obtain sequencing libraries, a two-step approach was applied. First, cDNA carrying full-length transcript information was synthesized with SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (TaKaRa Bio, Japan) followed by Nextera XT DNA Library Preparation Kit (Illumina, USA) according to the manufacturer's guidelines.