RNA-seq of Nicotiana tabacum
Firstly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperatures in an Illumina proprietary fragmentation buffer. The first strand of cDNA was synthesized using random oligonucleotides and Super Script II. The second strand of cDNA was synthesized using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities, and the enzymes were removed. After adenylation of the 3 ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with adaptor molecules ligated on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA).