Nicotiana tabacum L. cv. SR1 stem mRNA 5 developmental stages
Briefly, mRNA was purified from 20 µg of total RNA (a mixture of RNA from 5 wild type tobacco tissue samples and 3 35S::PtSEP3 tobacco tissue samples at equal ratio) using oligo (dT) magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperature. Taking these short fragments as templates, first-strand cDNA was synthesized using reverse transcriptase and random primers. Second-strand cDNA synthesis was followed using DNA polymerase? and RNaseH. Sequencing adapters were ligated to short fragments after purifying with QiaQuick PCR extraction kit, which were used to distinguish different sequencing samples. Fragments with lengths ranging from 200 to 700 bp were then separated by agarose gel electrophoresis and selected for PCR amplification as sequencing templates. The final cDNA library was sequenced using Illumina HiSeq™ 2000.