RNA-Seq of Tiftuf_Control leaf sample
Total RNA was extracted from Cynodon dactylon (bermudagrass) tissue samples and subjected to quality control to assess RNA integrity and purity. Messenger RNA (mRNA) was isolated using poly-T oligo-attached magnetic beads to selectively enrich polyadenylated transcripts. The purified mRNA was fragmented and used as a template for first-strand cDNA synthesis using random hexamer primers. Second-strand cDNA synthesis was then performed, incorporating dUTP or dTTP depending on whether a directional or non-directional library was desired.For non-directional libraries, cDNA was subjected to end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification.The final libraries were quantified using Qubit fluorometric quantitation and real-time PCR, and fragment size distribution was analyzed using a Bioanalyzer. Qualified libraries were pooled in equimolar concentrations and sequenced on an Illumina high-throughput platform based on the desired sequencing depth and effective library concentration.