Tetraodon embryos were obtained by in vitro fertilization of eggs and their development was observed through brightfield microscopy. The embryo development was divided into distinct stages using morphological features as described for normal development of other fish species like medaka (Oryzias latipes), zebrafish (Danio rerio), and fugu (Takifugu rubripes). Total RNA was extracted with Trizol (Invitrogen) according to the manufacturer s protocol from eggs, whole embryo at 30% epiboly (30 epi) and whole embryo at 24 hours post fertilisation (24 hpf). The RNA samples were treated with 2U Dnase I (Qiagen) per µg RNA sample at 37°C for 10 minutes. Digested samples were then treated with 20 mg/mL proteinase K (Sigma Aldrich) at 37°C for 45 minutes. The quality and quantity of total RNA were assessed with the Bioanalyzer 2100 (Agilent) and no sign of degradation was detected (RIN > 9.0). Sequencing libraries were generated from total RNA samples following the Truseq RNA protocol (Illumina). Single end reads (1 x 50 nucleotides) were obtained from 3 lanes on a Hiseq1000 using SBS v3 kits (Illumina).