title: RNA-Seq of Arundo donax: Ecotype A
design: The fifty-four total RNA samples from the three A. donax ecotypes were used for the construction of cDNA libraries and for Illumina sequencing reactions. Total RNA integrity was further checked using a Caliper GX (PerkinElmer, Waltham, MA). 1.5 µg of good quality RNA was used as input of the ‘TruSeq Stranded mRNA Sample Prep kit’ (Illumina, San Diego, CA) for library preparation following the manufacturer’s instructions. Briefly, the poly-A containing mRNA in each of the samples was purified and enriched using magnetic oligo(dT)-rich beads, and then fragmented using divalent cations under high temperature. Next, cDNA was synthesized by reverse transcription and standard blunt-ending plus add ‘A’. Illumina TruSeq adapters with indexes were ligated to the ends of the cDNA fragments. After ligation reaction and separation of non-ligated adapters, samples were amplified by PCR to selectively enrich the cDNA fragments in the library that have adapters at both ends. To ensure that the quality of the libraries was appropriate for sequencing, the concentration and insert size of final libraries were determined using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and Agilent 2100 Bioanalyzer High Sensitivity and DNA 1000 assay (Agilent Technologies, Santa Clara, CA). Finally, cDNA libraries were processed with Illumina cBot for cluster generation on the flowcell following the manufacturer’s instructions and sequenced in single-end mode using a HiSeq2500 sequencing platform (Illumina, San Diego, CA). The CASAVA v1.8.2 of the Illumina pipeline was used to process raw data for format conversion and de-multiplexing.