Transcriptome of S. aemula
RNA was extracted from leaves, flower buds, and mature flowers. We isolated RNA using either Trizol reagent, RNeasy kits, or a combination of the two methods. In the combined approach, the standard Trizol protocol was followed through the chloroform extraction step at which point 0.53X volumes of 100% ethanol was added to the aqueous phase and the entire RNA/ethanol mixture then applied to an RNeasy column and the Qiagen protocol followed thereafter. Approximately equal amounts of total RNA isolated from each tissue type were pooled prior to EST library preparation. For 454 sequencing , we used modified oligo-dT primers during cDNA synthesis to reduce the length of mononucleotide runs associated with the polyA tail of mRNA. Mononucleotide runs reduce sequence quality and quantity due to excessive light production and crosstalk between neighboring cells. For cDNA synthesis of the 454 libraries, we either used a broken chain short oligo-dT primer or two different modified oligo-dT primers: one to prime the polyA tail of mRNA during first strand cDNA synthesis and another to further break down the stretches of polyA sequence during second strand cDNA synthesis. We then normalized and amplified the cDNA using the TRIMMER-DIRECT cDNA Normalization Kit as above. After normalization, cDNA was sonicated to 500 to 800bp fragments and size-selected to remove small fragments. Then the fragmented ends were polished and ligated with adaptors. The optimal ligation products were selectively amplified and subjected to two rounds of size selection including gel electrophoresis and AMpure SPRI bead purification. Raw reads for each newly sequenced transcriptome were cleaned and assembled into contigs. We used the SnoWhite cleaning pipeline to remove adapters, chimeras, and other possible contaminating sequences from raw reads. The cleaned reads were assembled using MIRA with the recommended settings for Sanger or 454 assemblies. Following MIRA, we finished the transcriptome assemblies with CAP3 using a percent overlap of 94%.