Rneasy plant mini kit

Total RNA was extracted from taro leaf samples in RNAlater^® using the RNeasy Plant Mini Kit and synthesis of cDNA was performed using M-MLV reverse transcription (Promega, United States; Wang et al., 2017 (link)). A universal potyvirus-specific ELISA (Agdia Inc., USA) and a universal potyvirus-specific RT-PCR (Zheng et al., 2010 (link)) were used to test two samples with feathery mottling and distorted leaves, and one asymptomatic sample. RT-PCR with dasheen mosaic virus (DsMV) CI-specific primers and a DsMV-specific triple-antibody sandwich ELISA (Agdia Inc., United States; Wang et al., 2017 (link)) were used to confirm DsMV in taro leaf samples.
We used the CTAB method to isolate total DNA from 100 mg banana leaf samples with banana bunchy top virus (BBTV) symptoms kept in RNAlater^® using CTAB method and were tested by PCR using BBTV-Rep gene-specific primers and CP-specific primers BBTV-HAF1 and BBTV-HAR1 (Hamim et al., 2017 (link)). Six samples were also tested for BBTV using a BBTV-specific antibody in a triple-antibody sandwich-ELISA (Agdia, Elkhart, Inc.).

Three biological replicates of Arabidopsis thaliana Col-0 and Lp2-2 root segments were sampled after 3 days on SIM following protocol b (~100 mg fresh weight/sample). RNA extraction was done with the Qiagen RNeasy Plant Mini Kit and cDNA synthesis was done with the Promega GoScript^TM system using random hexamer primers. Yield and purity were assessed by nanodrop. WUS expression was normalized to UBC9 and TIP41L levels and a sample maximization strategy was applied for the plate layout (assays were spread over three runs, including two technical replicates, no-RT controls, and triplicate no-template controls). RT-qPCR was set-up with the GoTaq® master mix using primers described in Supplementary Table 1 and run on a Stratagene Mx3005P cycler. Results were analysed according to the ∆∆Ct method using the R package pcr^{98 (link)} (Supplementary Code).

RNA was isolated from purified haustoria and spores germinated for 15 h on sterile distilled water (16°C in the dark). Samples were ground to a fine powder in liquid nitrogen and total RNA isolated using the RNeasy Plant Mini Kit (Qiagen). Extracted RNA was treated with RNase-free DNAse (Promega) and repurified using the RNeasy Plant Mini Kit columns. RNA quality was assessed with the Bioanalyzer 2100. About 10 μg of total RNA was processed with the mRNA-Seq Sample Preparation kit from Illumina to produce the sequencing libraries. Quality and quantity controls were run on an Agilent 2100 Bioanalyzer using a DNA 1000 chip kit and each library was diluted and used for sequencing with an Illumina Genome Analyser GX II platform (100 bp paired-end reads).