RNA-seq of barley segregants resistant to BaYMV
Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (New England Biolabs, USA) following manufacturers recommendations. Briefly, 1.5 g of total RNAs per sample was used as input materials for purifying mRNA using poly-T oligo-attached magnetic beads. The purified mRNAs were fragmented and then subjected for cDNA synthesis, overhangs blunting, adenylation and ligation with NEBNext adaptors. The cDNA fragments with right length were purified using AMPure XP system (Beckman Coulter, Beverly, USA), and then subjected for pre-treatment using the USER Enzyme and following PCR reaction using Phusion High-Fidelity DNA polymerase. The PCR products were purified with AMPure XP system and the library quality was assessed using Agilent Bio-analyzer 2100. After cluster generation, the libraries were sequenced using Illumina Nova-seq S6000 platform (Illumina, USA) and 150 bp paired-end reads were generated.