GSM9573694: Shield, CapTrap-seq SS500; Danio rerio; RNA-Seq
Adult AB wild-type zebrafish (> 3 months old) were first anesthetized and subsequently euthanized using Tricaine methanesulfonate (MS-222) (Sigma Aldrich, Catalog No. A5040-25G) solution before being used for heart and testis dissection. Heart and testis tissues, as well as zebrafish embryos at the desired developmental stages, were washed in 1× PBS to remove contaminants and then homogenized in TRIzol reagent. RNA was extracted via isopropanol precipitation, followed by treatment with RNase-Free DNase (Qiagen, Cat. No. 79254) to eliminate genomic DNA contamination, and subsequently purified using Agencourt RNAClean XP beads (Beckman Coulter, Cat. No. A63987).  RNA samples (RIN ≥ 8.5) were pooled from 1,000 embryos per developmental stage, from 50 adult zebrafish (25 females and 25 males) for heart tissue, and from 50 males for testis tissue. The pooled RNA was concentrated using sodium acetate–ethanol precipitation. RNA concentration and purity were assessed using a Quantus Fluorometer (Promega) and a Nanodrop Spectrophotometer (Thermo Fisher Scientific), respectively, while RNA integrity was verified using the 2200 TapeStation system (Agilent Technologies). Potential genomic DNA contamination was evaluated by PCR amplification, and the RNA samples were stored at –80°C until further use. The CapTrap-seq cDNA libraries for the 2–4 cell stage, 28 hpf, heart, and testis were size-selected using the ProNex® Size-Selective Purification System (Promega, Catalog No. NG2001) to enrich for cDNA fragments longer than 500 bp. For each sample, 2 µg of the cDNA library was mixed with ProNex beads at a 1:1.1 ratio to selectively retain cDNA molecules exceeding 500 bp, followed by purification and elution according to the manufacturer's protocol.The size-selected fraction was assessed for quality using an Agilent 2100 Bioanalyzer and quantified with a Qubit fluorometer. Samples for Oxford Nanopore Technologies (ONT) MinION long-read sequencing were prepared using the Nanopore Ligation Sequencing Kit (SQK-LSK109), following the manufacturer's protocol (version ACDE_9064_v109_revG_23May2018). To achieve optimal loading concentrations for MinION sequencing, 500 ng of each cDNA library was used as input material. A total of 150 fmol of each Nanopore sequencing library was loaded onto separate R9.4.1 flow cells (FLO-MIN106D) and sequenced for at least 72 hours.