title: GSM8491565: Endothelium, Hpa2 loss-of-function, biol rep 1_2; Danio rerio; RNA-Seq
library_construction_protocol: 60-80 Tg(fli1a:eGFP)y1 Hpa2-loss of function or control larvae were anesthetized and euthanized by icing at 96 hpf. Larvae were washed once with PBS and subsequently dissociated into single cells using a 0.25% (w/v) trypsin 0.53 mmol/l EDTA solution at 30°C, applying gentle mechanical stress by pipetting. The reaction was stopped by adding twice the volume of 10% (v/v) calf serum/DMEM medium followed by two washing steps with PBS (cell pelleting 500x g for 5 min). For FACS sorting, cells were resuspended in sorting buffer (5mmol/l EDTA, 1% (v/v) goat serum in HBSS) and filtered through a 40-µm nylon mesh. Propidium iodide (1 µg/ml) was added directly prior to sorting of GFP+ cells. Sorted cells were kept on ice and RNA was isolated with the use of the RNeasy Plus Micro Kit (Qiagen). Library construction and sequencing were conducted by Novogene with the ultra low input pipeline for mRNA sequencing