PacBio Iso-seq of zebrafish: high
Total RNA was isolated from each developmental stages of zebrafish embryos using TRIzolTM Reagent (Invitrogen). RNA purity and concentration were assessed with the NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and the Qubit RNA Assay Kit in the Qubit 3.0 Fluorometer (Life Technologies, CA, USA). The RNA integrity number (RIN) was determined using the RNA Nano 6000 Assay Kit and Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA samples with a RIN  8 were used to synthesize cDNA with SMARTerPCR cDNA Synthesis Kit (Takara Bio USA, Inc., Mountain View, CA, USA). PCR amplification was performed using a KAPA HiFi PCR Kit (Kapa Biosystems, Wilmington, MA, USA) with the optimized number of cycles. Size selection of PCR products (cDNA) for each sample was applied using the BluePippin System: <3 kb and >3 kb. Subsequently, two cDNA libraries (<3 kb, >3 kb) were prepared using a SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, CA, USA) and sequenced on the PacBio sequel II platform.