GSM9737578: muscle-MA2; Carassius auratus; RNA-Seq
Total RNA was extracted from the tissue samples. RNA concentration and purity were then evaluated. RNA integrity was rigorously assessed using RNA-specific agarose gel electrophoresis and the Agilent 2100 Bioanalyzer. Poly(A) mRNA was enriched from total RNA using Oligo(dT) magnetic beads and subsequently fragmented into approximately 300 bp pieces using ion fragmentation. First-strand cDNA was synthesized using 6-base random primers and reverse transcriptase, followed by second-strand cDNA synthesis. After initial library construction, PCR amplification was performed to enrich the fragments, and size selection was conducted to yield a final library size of approximately 450 bp. Library quality, including total and effective concentrations, was rigorously assessed using an Agilent 2100 Bioanalyzer. Libraries containing different index sequences were then pooled proportionally based on their effective concentration and target data volume. The pooled libraries were diluted to 2 nM and denatured into single-stranded DNA via alkaline treatment. Finally, Next-Generation Sequencing (NGS) was performed on an Illumina platform to generate paired-end (PE) reads.