Trimmer kit

Total RNA was isolated from each of the fragments from the different treatments described above using TRIzol (Invitrogen) according to manufacturer’s instructions. The quality of all of the RNA was checked using a Bioanalyzer (Agilent) RIN ≥9.5, and pools with the same amount of RNA were then created. The cDNA library was constructed using a Clontech SMARTer PCR cDNA synthesis kit and amplified using the Advantage 2 PCR kit according to the manufacturer's instructions. Subsequently, 2 µg of the amplified cDNA was normalized using the Trimmer kit (Evrogen) following the manufacturer's instructions and purified using the Qiaquick PCR purification kit (Qiagen). The normalized and non-normalized cDNA was sent to Roche for further analysis. Sequencing was performed using a 454 GS-Flx instrument according to the manufacturers' instructions (Roche). In order to obtain the abundant and rare transcripts, the library placed on the 454 plate was divided into two, half containing normalized cDNA, and the other half with non-normalized cDNA. The normalized and non-normalized cDNAs were sheared by sonication to produce short random fragments (300–400 bp) appropriate for 454 sequencing, and oligonucleotide adaptors were then ligated to the fragmented sequences. The 454 GS-Flx running plate was divided for internal comparison of normalized and non-normalized libraries (data not shown).

cDNA normalization was performed from total RNAs with MINT and TRIMMER kits from Evrogen according to the manufacturer’s instruction, except that the number of PCR cycles for material amplification was adapted to our material. First, full length double stranded (ds) cDNA were synthetized from 2 μg of total RNA using the MINT kit [55 (link)]. First strand was synthetized from a fusion primer containing an oligo (dT) stretch to anneal RNA polyA tails. A poly (dC) stretch was incorporated at the end of the first strand, and used for priming the synthesis of the second strand. Full length (ds) cDNA were subsequently amplified by PCR, purified on Qiaquick columns (Qiagen) and checked for quality and yield before normalization. Normalization was done with the TRIMMER kit (Evrogen) which is based on DSN technology [56 (link)]. The method involves denaturation-reassociation of cDNA, Duplex Specific Nuclease (DSN) degradation of the ds-fraction corresponding to abundant transcripts and PCR amplification of the single strand (ss) DNA fraction. We started from 600 ng (ds) cDNA for normalization and after denaturation, incubated samples at 68°C for five hours for renaturation. After degradation of (ds) complexes by DSN, we made two runs of PCR amplification for optimal recovery. Normalized cDNA was then purified on Qiaquick columns (Qiagen) and yield was measured by spectrophotometry.

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.