Illumina HiSeq 2500 sequencing; Single-cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish
Single-cell transcriptome analysis of gata5GFP labelled WT and Gata5/6 deficient cells from early gastrulation to early somitogenesis in zebrafish
WT and Gata5/6KD TgBAC (gata5: EGFP) embryos were synchronized and dechorionated with pronase (Sigma, Cat# 11459643001) at desired stages. For embryos at gastrulation stages (6 hpf, 8 hpf, 10 hpf), the dissociation was performed by incubating embryos in 200 μl calcium-free Ringer solution (116 mM NaCl, 2.6 mM KCl, 5 mM HEPE, pH 7.0) for 5 min followed by 500ul TrpLE (GIBCO, TrypLE Express Enzyme, cat #: 12604-013) at room temperature. Dissociation was monitored under a dissecting scope and embryos were homogenized every 5min until single-cell suspensions were generated. Embryos at the early segmentation stage (13 hpf) were treated similarly but incubated with 500 ul of 0.25 Trypsin (GIBCO, Trypsin(2.5%), no phenol red, cat#: 15090046)/ EDTA instead of TrpLE for dissociation. After dissociation, Cells were washed twice with DMEM 1% FBS. DAPI was added at a concentration of 5ug/ml to exclude dead cells right before the Fluorescence-activated cell sorting (FACS). FACS was performed on Sony SH800S Cell Sorter, MoFlo XDP or MoFlo Astrios with a 100 μm nozzle by the SickKids-UHN Flow and Mass Cytometry Facility. Around 20,000 to 50,000 gata5GFP+ cells were obtained in one FACS experiment. Single cell suspensions after FACS were loaded into the 10x machine and RNA was extracted based on Chromium Single Cell 3' Reagent Kits v2 protocol. Libraries were constructed using the 10x v2 chemistry.