RNA-Seq of Camellia oleifera: C36h
Total RNA of each sample was extracted three times using a CTAB procedure. Leaves and roots of each sample were extracted separately and mixed with equal amounts of mRNA after examination. The RNA samples were dissolved in 10 mM Tris (PH 7.6) and examined using the NanoDrop ND-8000; the A260/A280 ratios of both samples ranged from 1.9 to 2.1. The integrity of the RNA samples was assessed with an Agilent 2100 Bioanalyzer; no sign of degradation was found.For the synthesis of cDNA and Solexa sequencing, we prepared 45 ŚĚg of total RNA for treated and control sample at concentrations of approximately 1500 ng/ŚĚl. We then enriched the poly (A) mRNA using beads with Oligo (dT) and interrupted mRNA into short fragments with fragmentation buffer. Using these short fragments as templates, we synthesized first-strand cDNA with hexamer-primers and reverse transcriptase (Invitrogen). The second-strand cDNA was synthesized using buffer, dNTPs, RNaseH (Invitrogen) and DNA polymerase I (New England BioLabs). The short fragments were then purified using a QiaQuick PCR extraction kit and re- solved with EB buffer to finish the end reparation, and were connected using sequencing adaptors. After reso- lution by agarose gel electrophoresis, we selected frag- ments suitable for PCR amplification. We then constructed two paired-end libraries which were se- quenced using an Illumina HiSeqTM 2500.