GSM5851880: Mock inoculated - 1 hpi - replicate 2; Malus domestica; RNA-Seq
Total RNA was extracted by adding 800 µL of CTAB extraction buffer (2 % CTAB, 2 % PVP40, 100 mM Tris-HCl (pH8), 25 mM EDTA, 2 M NaCl, 0.5 g L-1 spermidine, 1 % β-mercaptoethanol) to 150-200 mg finely crushed tissue sample. Subsequently, 80 µL N-laurylsarcosine sodium salt (20 %) and a glass bead were added, and the mixture was incubated for 10 min in a thermomixer set at 70 °C and 1400 rpm. Next, 900 µL chloroform:isoamylalcohol (24:1) was added and the samples were centrifuged at 21500 G and 22 °C for 10 min. The supernatant was transferred to the gDNA eliminator spin column of the RNeasy Plus Mini kit from Qiagen (Manchester, UK). From here on, the manufacturer's guidelines for the kit were followed. Quality and concentration of the extracts were verified using a NanodropTM2000 (Leusden, Netherlands) and by gel electrophoresis. Equal amounts of RNA were pooled across experiments to obtain three unique replicates per treatment per time point. The pooled RNA was precipitated by adding 2 vol 100 % ethanol and 0.1 vol 3 M sodium acetate (pH5.2-5.5), incubating at -80 °C overnight, and centrifugating at 21500 G. The precipitate was washed twice with 70 % ethanol before being resuspended in nuclease free water. All A260/A280 and A260/A230 ratios were above 2. A stranded library was constructed for each sample with fragment lengths of 75 bp, as described by Zhong and coworkers (Zhong et al., 2011). Sequencing was performed on an Illumina Nextseq500 platform (San Diego, USA). Generated reads were cleaned up and subsequently aligned to the 'Golden Delicious' apple genome (Daccord et al., 2017).