RNA-Seq of drought tolerant alfalfa
Middle leaflets from fully expanded trifoliate leaves (first from the shoot tip) were harvested from three biological replicate wild-type plants (N4.4.2 alfalfa), as well as three biological replicates of RNAi-205 and RNAi-207 genotypes, respectively, which had been grown under control (well-watered) and water-deficit (volumetric soil moisture of approximately 10%) conditions. Total RNA was extracted, RNA integrity was evaluated, libraries were prepared, and sequencing was carried out using an Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA) with 150-bp paired end reads through a service provider (Novogene Corporation Inc., Sacramento, CA) as described previously (Subedi et al., 2025). Raw read data were cleaned and trimmed using in-house perl scripts (Novogene, Sacramento, CA), and the resulting reads were mapped to the Medicago truncatula (v. 4.01.4) reference genome, which is a close diploid relative of alfalfa and has a very well-annotated genome sequence, using Hisat2 v2.0.5 (Kim et al., 2015) .