Normalized EST Library Generation

The same strain was used to generate a normalized EST library. The fungus was pre-cultivated in 50 ml of 2% malt broth (20 g l^-1 malt extract; Difco) for 14 days at 20 °C under constant shaking. Then, the mycelium was homogenized with a blender for 30 s and 5 ml of the homogenized mycelium was transferred to new 50 ml of 2% malt broth (20 g l^-1 malt extract; Hefe Schweiz). After 48 h the actively growing mycelium was harvested and immediately frozen in liquid nitrogen. Total RNA was isolated from approx. 75 mg fresh mycelium using the RNeasy plant mini kit (Qiagen, Hombrechtikon, Switzerland). Full-length cDNA was synthesized using the MINT kit (evrogen, Moscow, Russia) with a degenerated poly-T primer (5′-AAGCAGTGGTATCAACGCAGAGTAC (T)₄G(T)₉C(T)₁₀VN-3′) during the first strand cDNA synthesis [95 (link)] and polTM1 (5′-AAGCAGTGGTATCAACGCAGAGTACTTTTGTCTTTTGTTCTGTTTCTTTTVN-3′) for the generation of dsDNA. The cDNA was normalized using the TRIMMER kit (evrogen) and the library was sequenced on the 454. Resulting reads were filtered for chimeras and then a whole transcriptome assembly was performed in newbler 2.3 with a minimum overlap of 50 bases and 98% sequence similarity.