GSM5235246: RNA_seq_the other ECs; Danio rerio; RNA-Seq
Tails resected from TgBAC(isl1:TagRFP);Tg(dab2:EGFP) embryos at 48 hpf were subjected to mechanical and enzymatic dissociation by pipetting every 5 minutes in 1 ml of protease solution (TrypLE Express with 2.7 mg/ml Collagenase P) and incubated at 28 °C until full dissociation (~15 min). Cells were pelleted (3000 rpm, 5 minutes at 4 °C) and resuspended in suspension medium (phenol red free Dulbecco's modified Eagle's medium with 1% FBS and 0.8 mM calcium chloride) twice. Resuspended cells were passed through a cell strainer and subjected to cell sorting using a FACS Aria III cell sorter to isolate live TagRFP+/EGFP+ cells and TagRFP-/EGFP+ cells separately. For scRNA-seq, single cell suspensions were resuspended with the suspension medium and barcoded with a 10x Chromium Controller (10x Genomics). RNA from the barcoded cells for each sample was subsequently reverse-transcribed and sequencing libraries were constructed with reagents from a Chromium Single Cell v3.1 reagent kit (10x Genomics). Sequencing was performed with Illumina NovaSeq6000.