Hypericum perforatum L. flower transcriptome
Total RNA was extracted from whole flowers and flower parts by using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich) following the protocol provided by the manufacturer. The eventual contamination of genomic DNA was avoided by the optional DNase I (Sigma-Aldrich) treatment. Poly(A)+ mRNA was isolated from total RNA using the Ambion MicroPoly(A) Purist Kit according to the manufacturer’s instructions (Life Technologies, Darmstadt, Germany) and the integrity and quantity was verified using an Agilent 2100 Bioanalyser and RNA Nanochips (Agilent Technologies, Santa Clara, USA). In order to avoid over representation of the most commonly transcribed genes and to maximize sequence diversity, normalized, full-length enriched cDNA libraries were generated using a combination of the SMART (Zhu et al., 2001) cDNA library construction kit (Clontech, Takara, Saint-Germain-en-Laye, France) and the DSN normalization method (Zhulidov et al., 2004) implemented in the Trimmer Direct cDNA normalization kit (Evrogen, BioCat, Heidelberg, Germany). The procedure generally followed the manufacturer’s protocol but included several important modifications, essentially as previously described in more detail (Vogel et al., 2011). Optimization of the complete cDNA normalization procedure, such as the number of thermocycles for ds-cDNA synthesis, was essentially carried out as described by Vogel and Wheat (2011). Each of the resulting ds-cDNA pools was purified and concentrated using the DNA Clean and Concentrator kit (Zymogen) and size fractionated with SizeSep 400 spun columns (GE Healthcare) that resulted in a cutoff at ~200 bp. To test for normalization efficiency, a fraction of the full-length-enriched cDNAs were cut with SfiI and ligated to pDNR-Lib plasmid (Clontech). Ligations were transformed into E. coli ELECTROMAX DH5α-E electro-competent cells (Invitrogen). For each cDNA library, appr. 200 bacterial colonies were grown in 96 deep-well plates, plasmid minipreparations were performed using the 96well robot plasmid isolation kit (NextTec) on a Tecan Evo Freedom 150 robotic platform (Tecan) and Single-pass sequencing of the 5’ termini of cDNA libraries was carried out on an ABI 3730 xl automatic DNA sequencer (PE Applied Biosystems). Vector clipping, quality trimming and sequence assembly using stringent conditions (e.g. high quality sequence trimming parameters, 96% sequence identity cutoff, 35bp overlap) was done with the Lasergene software package (DNAStar Inc.). For direct sequencing, approximately 1 μg of each SfiI-digested normalized cDNA sample was sheared via nebulization into small fragments. The eight shotgun cDNA libraries were grouped in two plates and sequenced on the Roche 454 FLX platform using Titanium chemistry (Roche Diagnostics Corporation, Basel, Switzerland).