RNA-Seq of Tetrastigma hemsleyanum: fiber roots
Total RNA was extracted from Tetrastigma hemsleyanum roots using Trizol reagent. RNA purity and concentration were assessed by NanoDrop 2000 spectrophotometer, and RNA integrity was evaluated using Agilent 2100/LabChip GX. Quality-verified samples were used for library construction. For library preparation, mRNA was purified using magnetic beads with Oligo(dT) and fragmented in Fragmentation Buffer. First-strand and second-strand cDNA were synthesized using fragmented mRNA as template, followed by purification, end repair, poly(A) tailing, and adapter ligation. Size-selected fragments were PCR-amplified to generate cDNA libraries. Library quality was validated using Qsep400 for insert size determination and Q-PCR for effective concentration quantification (2 nM). Qualified libraries were sequenced on the Illumina NovaSeq 6000 platform using PE150 mode. Raw sequencing data in fastq format were filtered to remove adapters and low-quality reads (reads with >10% N content or >50% bases with Q10). Clean reads were mapped to the T. hemsleyanum reference genome (NCBI BioProject: PRJNA725161) using HISAT2, and transcripts were assembled with StringTie for downstream analysis.
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