Rneasy plant midi kit

Cadophora sp. DSE1049 and P. macrospinosa DSE2036 were maintained in modified Melin-Norkrans (MMN) liquid medium^{51 (link)} containing 3 g/L glucose and were grown as a free-living vegetative mycelium for two weeks at room temperature in the dark. For harvesting, the mycelium was dried on filter paper, flash-frozen in liquid nitrogen and ground into a powder. DNA was extracted from 2.5 g mycelium using the DNeasy Plant Maxi kit (Qiagen) according to the manufacturer’s instructions (doing on-column RNase treatment). In addition, total RNA was isolated from 0.5 g mycelium using the RNeasy Plant Midi Kit (Qiagen) according to the manufacturer’s instructions, including DNase treatment.

Total RNA was extracted from entire shoots, including leaves and traps, from all three sources described above. Inflorescences were not used in this study. Approximately 800 mg of fresh weight (2 individuals) of whole shoots in three replicates from each source were ground in liquid nitrogen using a mortar and pestle and RNA was extracted using RNeasy Plant Midi Kit (Qiagen Inc., Valencia, CA, USA). The results of the parallel extractions were pooled in equal proportions into a single sample and the quality and quantity of the extracted RNA were verified by gel electrophoresis and by spectrophotometer, measuring the 230/260 ratio with Biophotometer (Eppendorf, Germany).
A pooled RNA sample was provided to GATC Biotech (Konstanz, Germany) for the construction of a cDNA library and subsequent sequencing. Library construction involved DNAselection for polyadenylated (polyA+) transcripts to enrich for protein-coding mRNAs, DNase I treatment and normalization through denaturation/reassociation of cDNA, to improve the representation of low-copy transcripts and thereby maximize gene discovery. The resulting library was sequenced with a full picotiter plate on a 454 GS-FLX sequencer with Titanium reagents (Roche Applied Science, Indianapolis, IN, USA), using standard 454 protocols.