GSM9533671: pd1116_HP_11_S9; Danio rerio; RNA-Seq
Total RNA was extracted by adding 300 µL of TRIzol reagent and allowing the homogenate to thaw. Following the addition of 60 µL of chloroform and vigorous vortexing, the phases were separated using Phase Lock Gel Heavy tubes (QuantaBio) centrifuged at 12,000 rcf for 15 minutes at 4°C. The aqueous phase was recovered and further purified using the RNeasy MinElute Cleanup protocol (Qiagen). Briefly, the sample was mixed with 70% ethanol and transferred to an RNeasy MinElute spin column. The membrane was washed with Buffer RPE and 80% ethanol, followed by a 5-minute centrifugal drying step with open lids to eliminate residual ethanol. RNA was eluted in 14 µL of RNase-free water. Strand-specific RNA-seq libraries were constructed from total RNA. Poly-adenylated RNA was enriched using oligo(dT) beads and fragmented. First-strand cDNA was synthesized using random hexamer priming, followed by second-strand synthesis incorporating dUTP to preserve strand information. Libraries were sequenced on an Illumina platform.