Normalized cDNA transcriptome sequencing for Pteridium aquilinum subsp. aquilinum gametophytes
Total RNA was isolated using the Sigma Spectrum Plant Total RNA Kit, incorporating on-column DNase I (Qiagen) digestion during extraction to remove traces of genomic DNA. Total RNA was concentrated by precipitating in 2.5 M ammonium acetate and 70 % ethanol, then resolubilizing the RNA pellet in RNase-free water to approximately 500 ng/*L. Total RNA was quantified and its quality verified using an Agilent Bioanalyzer 2100. Total RNA was sent to the Center for Genomics and Bioinformatics at Indiana University, Bloomington (IU CGB), where a normalized transcriptome (cDNA) library optimized for Roche 454 GS-FLX Titanium sequencing was prepared (J Buchanan-Carter and K Mockaitis, unpublished, IU CGB). Briefly, full-length enriched cDNA was synthesized with the CloneTech SMART cDNA synthesis kit using modified 454-ready adapter/primer oligos (K Mockaitis, unpublished). The frequency of abundant cDNA species was reduced using the Evrogen Direct Trimmer normalization kit. Normalized cDNA was fragmented by sonication, blunt end repaired, and ligated to custom 454 sequencing adapters. Amplification of the sequencing library incorporated an adapter-mediated PCR suppression effect to preferentially amplify ligation products suitable for 454 sequencing. This library was sequenced using the standard GS-FLX Titanium protocol on 3 regions of a 4-region PicoTitre plate.