title: RNAseq of Salvia hispanica_UniMelb: Leaf
design: RNA-seq libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, MA, USA). Poly(A) enrichment and rRNA removal were performed with Oligo-d(T) magnetic beads (NEBNext Poly(A) mRNA Magnetic Isolation Module). Fragmentation was conducted with NEBNext First Strand Synthesis Reaction Buffer (5X), followed by priming with NEBNext Random Hexamer Primers. Second-strand synthesis was carried out with ProtoScript II Reverse Transcriptase and NEBNext Second Strand Synthesis Enzyme Mix (NEB). Double-stranded cDNA was purified with AxyPrep MAG PCR Clean-up Kit (Axygen, CA, USA), and end-repair was completed using NEBNext End Prep Enzyme Mix (NEB). A-tailing was done with the NEBNext dA-Tailing Module (NEB), and adaptor ligation was conducted using Illumina-compatible adaptors and DNA ligase, following the NEBNext Ultra RNA Library Prep Kit protocol. Adaptor-ligated cDNAs were size-selected using the AxyPrep MAG PCR Clean-up Kit (Axygen) to obtain fragments of approximately 360 bp (insert size ~300 bp). The libraries underwent PCR amplification (11 cycles), were validated using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and were quantified using a QubitTM 2.0 Fluorometer (Invitrogen, CA, USA). Sequencing was conducted on an Illumina HiSeq X-ten platform in 2150 bp paired-end mode, yielding 2,116,114,396 reads (~635 Gb) across 56 RNA-seq libraries.