De novo transcriptome sequencing in a desert tree, Caragana korshinskii Kom.
After Caragana korshinskii seedling grow for one month, the different tissues from seedlings, including leaves, stems and roots, were harvested together for RNA isolation.The total RNA of each sample was then pooled at equivalent quantities. Sequencing libraries were generated using a NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations. Following the manufacturer’s procedures, mRNA was purified from the pooled total RNA using polyT oligo-attached magnetic beads. A fragmentation buffer was added to disrupt the mRNA into short fragments. Reverse transcriptase and random primers were used to synthesise the first-strand cDNA from the cleaved mRNA fragments. The second-strand cDNA was synthesised using buffer, dNTPs, RNaseH, and DNA polymeraseI. The double-strand cDNA was purified using the QIAquick PCR extraction kit (QIAGEN, Hilden, Germany) and washed with EB buffer for end repair and single nucleotide A (adenine) addition. Finally, sequencing adaptors were ligated onto the fragments. The required fragments were purified by AMPure XP beads and enriched by PCR to construct a library for transcriptome sequencing.