GSM8898910: Heart, TSO; 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. Template Switching Oligo (TSO): 5 μg of total RNA per sample (heart, testis, 2–4 cell, 28 hpf) was combined with 3 μl of a 1:100-diluted, pre-5′-capped SIRV-Set 4 (Lexogen) and processed for rRNA depletion using the riboPOOL kit for Danio rerio (SiTools, Cat. No. 27DP-K012-000010) following the manufacturer's protocol (riboPOOLKitManual_v6). After rRNA depletion, RNA was purified using Agencourt RNAClean XP beads (1.8× ratio) according to the manufacturer's recommendations.For cDNA synthesis, 4 μL of ribo-depleted RNA per reaction (performed in duplicates) was used for first-strand cDNA synthesis with Template Switching RT Enzyme Mix (NEB, Cat. No. M0466) and anchored oligo-dT, following the manufacturer's protocol. The resulting single-stranded cDNA underwent second-strand synthesis and long-range PCR (LA-PCR) using TaKaRa LA Taq (Takara, Cat. No. RR002M) with 9 PCR cycles. To minimize PCR duplicates, cDNA was split into two independent PCR reactions. PCR replicates were pooled, purified using AMPure XP beads (1× ratio), and resuspended in RNase-free water. Samples were quantified using a Qubit 4 Fluorometer and assessed for quality using an Agilent 2100 Bioanalyzer. For Oxford Nanopore Technologies (ONT) MinION long-read sequencing, libraries 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. CapTrap-seq: CapTrap‐seq cDNA libraries were prepared as described (Carbonell-Sala et al., 2024). 5 μg of total RNA per sample (2-4Cell, 28hpf, heart and testis) was combined with 3 μl of a 1:100-diluted, pre-5′-capped SIRV-Set 4 (Lexogen) and reverse-transcribed using PrimeScript II RTase (200 U/μl) and an anchored oligo(dT) primer. Full-length, 5′-capped transcripts were enriched via the captrapper method. Briefly, the 5′ cap structure was oxidized to introduce aldehyde groups, which were then biotinylated to enable selective enrichment of capped RNA molecules with streptavidin-coated magnetic beads. Then specific adaptors were ligated to both cDNA ends to facilitate amplification, sequencing, and further enrichment of full-length molecules. The cDNA was converted into double-stranded form using 2× HiFi KAPA mix (Kapa, Cat. No. 7958927001-KK2601) and amplified by LA-PCR with TaKaRa LA Taq (Cat. No. RR002M, Takara) with 16 PCR cycles. Libraries were quantified using a Qubit 4 Fluorometer and quality-checked with an Agilent 2100 Bioanalyzer. For Oxford Nanopore MinION sequencing, libraries 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, 500 ng of each cDNA library was used as input, and 150 fmol was loaded onto separate R.9.4.1 flow cells (FLO-MIN106D) for a minimum of 72 hours of sequencing. CapTrap-seq SS500: 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.
GEO
GSM8898910_r1