RNA-Seq  of Medicago sativa: leaves at the seedling stage
First, mRNA was isolated from total RNA using Oligo(dT)-coated magnetic beads via A-T base pairing with poly(A) tails. Second, the purified mRNA was randomly fragmented into approximately 300 bp fragments by adding fragmentation buffer under optimized conditions. Third, first-strand cDNA was synthesized by reverse transcriptase using random primers, followed by second-strand synthesis to generate stable double-stranded cDNA. Subsequently, an End Repair Mix was added to blunt the ends of the cDNA, and an adenine (A) base was then added to the 3' ends to facilitate adapter ligation. Finally, the adapter-ligated products were purified and size-selected, and the resulting fragments were amplified by PCR to obtain the final sequencing library.