GSM9531978: TRBC28.9_Traffic road (Barcelona, cold season); Danio rerio; RNA-Seq
Organic molecular compounds were  extracted from DGI filter a with a 10 mL mixture of dichloromethane: methanol (2:1 v/v) has been already described (Ricarte et al., 2025). Extracts were finally resuspended in methanol and stored at -20ºC. RNA isolation, library preparation and RNA sequencing Total RNA was isolated from the pools of 10 zebrafish eleutheroembryos using TRizol (Invitrogen Life Technologies, Carlsbad, CA, USA) as described by the manufacturer. Extracted RNA was reconstituted in RNase-free water and assayed for quantity and quality using Qubit® RNA BR Assay kit (Thermo Fisher Scientific) and RNA 6000 Nano Assay on a Bioanalyzer 2100 (Agilent Technologies). Three high-quality RNA biological replicates per condition were sent for sequencing to the National Center for Genomic Analysis (CNAG, Barcelona, Spain). In all cases, RNA concentrations ranged between 50-200 ng·µL-1, free of DNA and with RNA integrity number (RIN) > 8.  Libraries for RNA-Seq were prepared from total RNA using KAPA Stranded mRNA-Seq Kit Illumina® Platforms (Kapa Biosystems) with minor modifications. Briefly, after poly-A based mRNA enrichment with oligo-dT magnetic beads and 500 ng of total RNA as the input material, the mRNA was fragmented (resulting RNA fragment size was 80-250nt, with the major peak at 130nt). The second strand cDNA synthesis was performed in the presence of dUTP instead of dTTP, to ensure the strand specificity. The blunt-ended double-stranded cDNA was 3´adenylated and Illumina indexed adapters (Illumina) were added by ligation. The ligation product was enriched through 15 PCR cycles, and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay.  Each library was sequenced using TruSeq SBS Kit v3-HS, in paired-end mode with a read length of 2x76bp. We generated on average 40 million paired-end reads for each sample in a fraction of a sequencing lane on HiSeq2000 (Illumina) following the manufacturer's protocol.