RNA-Seq of Secale cereale: seedlings infected with Microdochium nivale, 20 d.p.i.
Plant material (stems taken from infected plants with moderate disease manifestation) was ground in liquid nitrogen. The obtained powder was resuspended in 1 mL of ExtractRNA Reagent (Evrogen, Moscow, Russia). Residual DNA was eliminated using a DNA-free kit (Life Technologies, Carlsbad, California, USA). For RNA-Seq, total RNA (1 g) was enriched with mRNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Hitchin, UK). mRNA was processed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, Hitchin, UK) according to manufacturers instruction. The quality and quantity of the cDNA libraries before sequencing were monitored using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, California, USA) and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA). Libraries were sequenced in three biological replicates. Sequencing was conducted on Illumina HiSeq 2500 (Illumina, San Diego, California, USA) at the Joint KFURiken Laboratory, Kazan Federal University (Kazan, Russia).