GSM9616082: 52hpf; Danio rerio; RNA-Seq
Female prop1:GAL4;UAS:mScarlet-CAAX zebrafish were crossed with wild-type (AB) male zebrafish on consecutive days (24 hours difference), using separators to control mating times. At 28 hpf and 52 hpf embryos were placed on ice to stop their development and Tricaine was used as anaesthetic. Dissection was carried out manually, using insulin needles and a glass plate under a fluorescence microscope (Nikon SMZ18). The dissection for 28 hpf embryos consisted of removing most of the torso, while dissection of embryos at 52 hpf consisted in separating the head. Samples were collected and conserved in chilled 1X PBS until their disaggregation.    The single cell suspension was filtered through a Cell Strainer Snap Cap (Corning) and kept on ice. Before FACS, 1 uL of 1 mg/ml DAPI was added. Dissected sibling embryos that did not exhibit fluorescence were used as a negative sorting control. FACS sorting was performed on a Bigfoot spectral cell sorter (Thermo Fisher) and cells were captured in a minimal volume (20 µL) of PBS with 5% FBS in a 1.5 mL eppendorf tube, previously coated with PBS + 20% FBS. The collected cells were kept on ice until sequencing. ScRNA libraries were prepared using two lanes in a Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics) following the standardized protocol and the sequencing was performed on a NextSeq 2000 sequencer (Illumina). Raw reads were aligned using Cellranger 9.0.1 against the GRCZ11 reference. DNA from the transgenic lines bns714 and bns681 was added using the mkref package from CellRanger (10x Genomics).