GSM5941829: 5dpi_4; Malus domestica; Venturia inaequalis; RNA-Seq
Total RNA was extracted using a Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA), and DNA removed following treatment with DNase I (InvitrogenTM, Thermo Fisher Scientific, MA, USA) according to the manufacturers' instructions. RNA concentration and purity was quantified using a Nanodrop, ND-1000 Spectrophotometer (NanoDrop Technologies, Rockland, DE). RNA integrity was assessed on the Agilent 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany) using an Agilent RNA 6000 Nano Kit in conjunction with the Agilent 2100 Bioanalyzer software according to the manufacturer's instructions. RNA samples with an RNA Integrity Number (RIN) value of 3.5 or more, were used for sequencing. Library preparation based on Illumina standards:After quality control, mRNA from eukaryotic organisms was enriched using oligo(dT) beads. mRNA was fragmented randomly by addings fragmentation buffer, then the cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom and secon-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA was completed through size selection and PCR enrichment.