SCI_3d_RNA-Seq
Total RNA was isolated and quality-checked using agarose gel electrophoresis and NanoDrop ND-1000. mRNA was enriched via NEB Next Poly(A) mRNA Magnetic Isolation Module (or rRNA-depleted using RiboZero Magnetic Gold Kit). Strand-specific RNA-seq libraries were constructed with KAPA Stranded RNA-Seq Library Prep Kit (Roche) using dUTP-based method. Key steps included RNA fragmentation, first-strand cDNA synthesis, dUTP incorporation, end repair/A-tailing, Illumina adapter ligation, and PCR amplification. Final libraries were validated by Agilent 2100 Bioanalyzer and quantified via qPCR before pooling for Illumina sequencing.