GSM9514961: 1 µM sodium arsenite, bio rep 3; Danio rerio; RNA-Seq
Total RNA was extracted from pooled zebrafish larvae (n = 10 per biological replicate per treatment) using TRIzol reagent (CAT# 15596018; Thermo Fisher Scientific), following the manufacturer's protocol. RNA concentration and purity were assessed using a Nanodrop Lite (CAT# ND-LITE; Thermo Fisher Scientific) spectrophotometer (A260/280 ratio between 1.8 and 2.2, A260/230 ≥ 1.8), and RNA integrity was qualitatively evaluated by 1% agarose gel electrophoresis. Qualified RNA samples were shipped on dry ice to Novogene Co., Ltd. (Beijing, China) for library construction, sequencing, and differential gene expression with functional analyses. At Novogene, RNA integrity was assessed using an Agilent 2100 Bioanalyzer. Samples met the following quality criteria: A260/280 ratio between 1.8 and 2.2, A260/230 ≥ 1.8, RNA Integrity Number (RIN) ≥ 5.8, smooth baseline, and total RNA yield ≥400 ng. Polyadenylated mRNA was enriched from total RNA using oligo(dT) magnetic beads and fragmented using a proprietary fragmentation buffer. First-strand cDNA synthesis was performed using random hexamer primers and reverse transcriptase, followed by second-strand synthesis using dNTPs, RNase H, and E. coli DNA polymerase I. The resulting double-stranded cDNA underwent end repair, A-tailing, ligation with Illumina-compatible adapters, size selection, and PCR amplification. Libraries were validated on an Agilent Bioanalyzer, quantified using a Qubit 2.0 fluorometer (Life Technologies), and further assessed by qPCR to ensure a minimum library activity of >2 nM. 