Transcriptomic Analysis of Zebrafish Larvae Exposed to Tire Wear Particle Leachates

For transcriptomic analysis, total RNA was extracted from pooled zebrafish larvae (80 per sample, three biological replicates per group) preserved in RNAlater™. High-quality RNA (RIN > 7.0), isolated using the RNeasy Mini Kit, was quantified and assessed for integrity. Ribosomal RNA was depleted prior to cDNA library construction using the TruSeq Stranded Total RNA Library Prep Gold Kit. The resulting libraries were quantified, size-validated, and sequenced using the Illumina NovaSeqX platform with 2×100 bp paired-end reads. Overall design: Adult wild-type zebrafish were acclimated under controlled laboratory conditions for three months prior to breeding. Fertilized embryos were collected and exposed to vehicle control or one of three concentrations of tire wear particle (TWP) leachates (TWP 250, 350, and 700) from 4 to 168 hours post-fertilization (hpf). Each group included 200 embryos and was tested in triplicate. At 72 hpf, larvae were transferred to clean medium with adjusted water quality parameters while maintaining consistent temperature and photoperiod. For transcriptomic analysis, 80 larvae per replicate were pooled, and total RNA was extracted from three biological replicates per group. RNA samples with RIN > 7.0 were used for cDNA library construction, followed by high-throughput sequencing using the Illumina NovaSeqX platform.