RNA-Seq of Nicotiana tabacum: T6_2
A total amount of 1 g RNA per sample was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I (NEB, Ipswich, MA, USA) and RNase H (NEB, Ipswich, MA, USA). The cDNA libraries were constructed by using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina Inc., San Diego, CA, USA) according to the manufacturers instructions. The library was prepared for Illumina sequencing by Shanghai OE Biotech. Co., Ltd., and was sequenced on the Illumina sequencing platform (Illumina HiSeq X Ten).