title: Theobroma cacao pistil transcriptome 1
design: Total RNA was pooled from 13 pistil tissue extractions and 400 ng (as determined by Quant-iT Ribogreen (Invitrogen) fluorometric assay) was used to prepare a custom sequencing library optimized for the Roche/454 GS-FLX Titanium platform (K. Mockaitis, unpublished).  Briefly, total RNA was used in a modified SMART (Clontech) first strand cDNA synthesis with primers customized for 454, and amplified 16 cycles.  Double-stranded cDNA was partially normalized by double-stranded nuclease (DSN) digestion using the Trimmer Direct kit (Evrogen) and then fragmented by nebulization.  Fragments were end-polished and ligated to customized adaptors using reagents from the GS-FLX Titanium General Library Preparation Kit (Roche), followed by a limited amplification using suppression PCR (Meyer et al., 2009) to selectively amplify properly ligated templates.  The final library was size selected after agarose gel electrophoresis into two fractions. Sublibrary A at 300-500 bp and sublibrary B at 500-900 bp were cleaned additionally using AMPure XP beads (Agencourt) at ratios of 0.7x and 0.5x respectively.  Each was titrated separately by emulsion PCR and sequenced on an XLR70 instrument according to the manufacturer (Roche/454).  Roche/454 and IU CGB custom adapter sequences were removed from the reads according to GS FLX System Software of the manufacturer (Roche/454 Sequencing) followed by ESTClean (https://sourceforge.net/projects/estclean/).