RNA-seq of Medicago sativa: tetraploid_USP_pollinated_flower
RNA (mRNA) was isolated using oligo-dT beads (Dynabeads mRNA Direct Micro Kit, Thermo Fisher Scientific) following the protocol for mRNA isolation from purified total RNA. The quality and quantity were verified by TapeStation (Agilent Technologies), for detecting possible contaminations from 18S and 28S sequences. Sequencing libraries were prepared from mRNA samples using Ion Total RNA-seq Kit v2 (Thermo Fisher Scientific), following the instruction provided by the manufacturer, which include three main phases of the mRNA fragmentation with Rnase III and purification, the hybridization and ligation of RNA, the reverse transcription and the final amplification of cDNA. The double-stranded barcoded cDNA libraries were eluted in 15 l of nuclease-free water and their concentration and size distribution were assessed through D1000 screen Tape (Agilent Tapestation), then normalized to get a molar concentration of 100pM, and pooled. The sequencing run was performed using Ion 540 Chip on the Ion Torrent S5 System (Thermo Fisher Scientific).