Transcriptome analyses of Medicago sativa under cold stress

The aboveground parts of the Medicago sativa were collected for RNA-Seq at T_0h (after cold acclimation), T_12h, T_24h, and T_36h (0 h, 12 h, 24 h, and 36 h after the temperature was maintained at 4 degree Celsius). Samples (0.2 g) from three biological replicates of each treatment were ground with liquid nitrogen for RNA extraction, and subsequently, the RNA samples were subjected to rigorous quality control measures. For accurate assessment of RNA integrity, an Agilent 2100 Bioanalyzer was used for quality control [22]. The library construction process was performed following the protocol described by Niu and Ma [23]. Initially, total RNA was randomly fragmented and utilized for the synthesis of first-strand cDNA using the M-MuLV reverse transcriptase system. Subsequently, the second strand of cDNA was synthesized employing DNA polymerase I and dNTPs. Then, AMPure XP beads were utilized to select cDNA fragments in the range of approximately 370-420 bp. The selected fragments were subjected to PCR amplification and purification using AMPure XP beads, ultimately yielding the library.