Comprehensive Transcriptome Analysis of Goldfish Embryos

Embryos were observed under microscopes, and developmental stages were determined according to the developmental stages of common goldfish [3 (link),7 (link)]. Embryos at three stages, i.e., zygote period, 14-Somite stage, and 35% optic vesicle closure (OVC) stage were collected and subjected directly to RNA preparation using Trizol reagent. Five embryos were used for each stage. Total RNA quality was determined by electrophoresis. RNA from the three developmental stages were mixed equivalently, before library construction and sequencing on the PacBio platform. The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the Clontech SMARTer PCR cDNA Synthesis Kit and the BluePippin Size Selection System protocol, as described by Pacific Biosciences (PN 100-092-800-03). For gene expression analysis, RNA from each stage of Ryukin and Celestial-Eye goldfish were subjected to library preparation and sequencing separately, according to the protocol. RNA-seq was performed on the HiSeq 2000 platform.