title: RNA seq of the Cibotium barometz: petiole
design: RNA extraction and library preparation Total RNA was extracted from retiole using a modified CTAB method. Tissues were ground in liquid nitrogen, and RNA was isolated using CTAB buffer, followed by LiCl precipitation and DNase I treatment to remove genomic DNA contamination. RNA quality and quantity were assessed using an Agilent 2100 Bioanalyzer and Qubit fluorometer, respectively. RNA-seq libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) following the manufacturer's instructions. Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads, fragmented, and reverse transcribed into cDNA. Second-strand cDNA synthesis was performed with dUTP to preserve strand specificity. The resulting double-stranded cDNA was end-repaired, A-tailed, and ligated with adapters. The libraries were then PCR-amplified and size-selected to obtain insert sizes of 250-300 bp. Sequencing RNA-seq libraries were sequenced on an Illumina NovaSeq 6000 platform using 150 bp paired-end reads.