title: RNA-seq of wheat: Chrysosporium pseudomerdarium treatment 2
design: Total RNA was extracted from 100 mg of leaves using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA), which enables the isolation of high-quality RNA. The procedure was supported by the use of Plant Isolation Aid (Thermo Fisher Scientific, Waltham, MA, USA) and followed the protocol outlined by Smoczynska et al. (2019). RNA integrity was assessed via electrophoresis on a 1% agarose gel (BioShop, Canada) prepared in 1xTBE buffer (BioShop, Canada), and RNA concentration was determined spectrophotometrically using a NanoDrop ND-1000 device (Thermo Fisher Scientific, Waltham, MA, USA). Total RNA extracted from wheat tissues was used to construct 15 RNA-seq libraries, representing three biological replicates for each of five experimental conditions (four fungal inoculations and one control). Library preparation and sequencing were outsourced to a commercial provider (Genomed S.A., Warsaw, Poland). Sequencing was carried out on an Illumina HiSeq X platform using paired-end 150 bp reads (PE150), with approximately 50 million read pairs generated per sample.