Nanodrop ultraviolet visible uv vis spectrophotometer

150 mg of bark tissue collected from young flushes of C. macrophylla or leaf discs (1 cm in diameter) from N. benthamiana was ground in liquid nitrogen, and total RNA was extracted using the Trizol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the procedure of the manufacturer. RNA extracts were resuspended in 100 µL of RNase-free water. RNA concentration was measured using a NanoDrop Ultraviolet–Visible (UV–Vis) Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA extracts were diluted with RNase-free water to adjust concentration to 100, 10 and 1 ng/µL. Aliquots were stored at −80 °C until use.

For RT-qPCR validation of the stranded mRNA-seq data, cDNA was generated from the RNA samples using the GoScript™ Reverse Transcription System (Promega) with Oligo(dT) primers following manufacturer’s instructions. Approximately 80 ng of the cDNA was used as template in 10 μL qPCR reactions (5 μL AzuraView™ GreenFast qPCR Blue Mix LR (Azure Genomics), 250 nM forward primer, 250 nM reverse primer). The sequences of primers used are provided in Supplementary Table S1. The reactions were carried out in CFX Connect™ Real-time PCR detection system (BIO-RAD) with cycling conditions of 95°C for 2 min followed by 39 cycles of 95°C for 5 s and 60°C for 28 s. Fold changes were calculated with gene specific PCR efficiency correction and two reference gene normalization as described (Hellemans et al., 2007 (link)).
To determine the virus titer of the T36 and T68-1 variants in the inoculated plants, one-step RT-qPCR was performed with primer/probe pairs specific to either T36 or T68-1 variants as described in Bergua et al. (2016) (link). RNA concentration was determined using a NanoDrop Ultraviolet–Visible (UV–Vis) Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Standard curves were prepared with 10-fold dilutions ranging from 10⁹ to 10⁵ copies of a target transcript according to the procedure described in Bergua et al. (2016) (link).