RNA-Seq of rye plants infected with M. nivale F00608 (bulk)
For RNA-Seq analysis, total RNA was extracted from M. nivale mycelium grown for 10 days submerged in LSM and then sustained in the presence or absence of rye extract for 24 h (for the comparative transcriptome analysis and for the identification of protein-encoding M. nivale genes), as well as from axenically grown rye plants (cultivar Ogonek) infected with M. nivale 20 days post infection (Gorshkov et al. 2020) (for the identification of the protein-encoding M. nivale genes). Fungal mycelium or plant material was ground in liquid nitrogen. Total RNA was extracted using the ExtractRNA Reagent (Evrogen, Moscow, Russia) according to the manufacturers instructions. Residual DNA was eliminated using a DNA-free kit (Life Technologies, Carlsbad, CA, USA). RNA quantity and quality (RNA Quality Number, RQN >7) were analyzed using an Implen nanophotometer (Implen, Germany) and a Qsep100 DNA Analyzer (Bioptic, New Taipei City, Taiwan), respectively. For RNA-Seq, total RNA (1 g) was enriched with mRNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, USA). Then, mRNA was processed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, USA) according to the manufacturers instructions. The quality and quantity of the cDNA libraries before sequencing 196 were monitored using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, 197 CA, USA), respectively. For the quantification of the cDNA libraries, the EVA Green I PCR-Kit (Synthol, Moscow, Russia) and primers for Illumina adapters (Evrogen, Moscow, Russia) were used; PhiX Control (Illumina, USA) was used as a concentration standard. Libraries were sequenced in three biological replicates. Sequencing was conducted on an Illumina HiSeq 2500 (Illumina, USA) at the Joint KFURiken Laboratory, Kazan Federal University (Kazan, Russia).