RNA-seq of barley cv. Conlon
A total amount of 3 g RNA per sample was used as input material for the RNA samplepreparations. Sequencing libraries were generated using NEBNext Ultra RNA LibraryPrep Kit for Illumina (NEB, USA) following manufacturers recommendations and indexcodes were added to attribute sequences to each sample. Briefly, mRNA was purified fromtotal RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried outusing divalent cations under elevated temperature in NEBNext First Strand SynthesisReaction Buffer5X. First strand cDNA was synthesized using random hexamer primerand M-MuLV Reverse TranscriptaseRNase H-. Second strand cDNA synthesis wassubsequently performed using DNA Polymerase I and RNase H. Remaining overhangswere converted into blunt ends via exonuclease/polymerase activities. After adenylation of3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated toprepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bpin length, the library fragments were purified with AMPure XP system (Beckman Coulter,Beverly, USA). Then 3 l USER Enzyme (NEB, USA) was used with size-selected,adaptor-ligated cDNA at 37C for 15 min followed by 5 min at 95 C before PCR. ThenPCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primersand Index (X) Primer. At last, PCR products were purified (AMPure XP system) andlibrary quality was assessed on the Agilent Bioanalyzer 2100 system.