Small RNA-Seq  of Medicago sativa: leaves at the seedling stage
First, total RNA was extracted from tissue samples, and adapters were ligated to both the 3 and 5 ends. Second, under the action of reverse transcriptase, first-strand cDNA was synthesized using random primers with the adapter-ligated RNA as template, followed by second-strand synthesis to generate stable double-stranded cDNA. Subsequently, PCR amplification (1112 cycles) was performed using sequencing primers to enrich the library concentration. According to the length distribution characteristics of miRNAs, target fragments were recovered by gel excision (6% Novex TBE PAGE gel, 1.0 mm, 10 wells). Finally, the library was sequenced on the NovaSeq X Plus platform.