Whole transcriptomic sequencing of zebrafish rx3 mutants during optic vesicle morphogenesis. Danio rerio

To identify genes regulated by Rx3 during optic vesicle morphogenesis, adult zebrafish carriers of a null rx3 mutation were mated. Before 13 hours post fertilization (hpf), the earliest time point at which optic vesicle evagination phenotypes could be reliably detected, offspring were phenotypically separated into pools comprising of mutants with an absence of optic vesicles or siblings exhibiting a wild-type phenotype. Three replicates of pooled RNA samples from 13 hpf eyeless mutants (rx3-/-) or phenotypically wild-type siblings (rx3+/+ or rx3+/-), and one replicate of 13 hpf wild-type zebrafish larva were collected for whole transcriptome sequencing. Overall design: Whole transcriptome sequencing (RNA-seq) was performed on zebrafish rx3-/- mutants, wild-type siblings and wild-type AB strains at 13 hpf

为鉴定视泡形态发生过程中受Rx3调控的基因，研究人员将携带功能缺失型rx3突变的成年斑马鱼进行交配。在受精后13小时（hours post fertilization，下文简称hpf，该时间点为可稳定检测到视泡外翻表型的最早时间）前，将子代按表型分为两组混合样本池：一组为缺失视泡的rx3-/-突变体样本池，另一组为表现为野生型表型的同窝幼鱼样本池。研究人员收集了3组生物学重复的13 hpf无眼突变体（rx3-/-）混合RNA样本、3组生物学重复的13 hpf表型野生型同窝幼鱼（rx3+/+或rx3+/-）混合RNA样本，以及1组生物学重复的13 hpf野生型AB品系斑马鱼幼鱼样本，用于全转录组测序。整体实验设计：对13 hpf的斑马鱼rx3-/-突变体、野生型同窝幼鱼以及野生型AB品系幼鱼开展全转录组测序（RNA-seq）。