Concatenated Stem RNA-Seq of Mentha x piperita (Mitcham peppermint) exposed to Verticillium wilt
GFP-labeled Verticillium dahliae was grown in Difco Czapek-dox broth (Becton, Dickinson and Company, Sparks, MD, USA) on a shaker at 200 RPM at room temperature for 2 weeks. The liquid medium (200 mL) was inoculated with three to four ~0.5 cm2 agar plugs from a V. dahliae Czapek-dox agar plate. On the day of inoculation, cultures were strained to remove mycelia and centrifuged at 10,000 g for 5 min pellet conidia. Conidia were resuspended in H2O to the  of the original volume of the culture. Counts were done under light microscope using hemocytometer. Conidia were diluted to a final concentration to about 107 conidia/mL. Briefly, rooted cuttings were removed from soil and cleaned from debris, and 23 plants were submerged into a beaker containing either water or conidial suspension and incubated for 5 min. Inoculated cuttings were randomly replanted in new flats in autoclaved soil and incubated in the growth chamber. Root tissue and stem tissue representing the first two internodes above the soil line were collected into separate tubes at four time points post-inoculation: 10 dpi, and 20 dpi. Tissue samples were immediately flash-frozen and stored at 80 C prior to RNA extraction. Both root and stem plant tissues were ground in liquid N2 using mortar and pestle and followed by RNA extraction protocol provided by Qiagen RNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA) with on-column DNA digestion protocol. The RNA concentration was estimated by Nanodrop (Thermo Fisher, Waltham, MA, USA), and quality assessment was done using a Bioanalyzer (Agilent, Santa Clara, CA, USA). Sequencing libraries were prepared and Illumina sequencing was performed at the Oregon State University Center for Genome Research and Biocomputing. Samples that showed amplification were additionally treated with DNase using DNaseMax kit (Qiagen Germantown, MD, USA). The cDNA was prepared from 1 g RNA using iScript RT Supermix for RT-qPCR (BioRad, Hercules, CA, USA), following manufacturers instructions. Primers efficiencies were more than 90%. qPCR was done in a reaction volume of 10 L containing 1 L of cDNA, 5 L of 2  SYBR SoAdvanced Univ SYBR Green Suprmix (BioRad, Hercules, CA, USA), and 0.5 L of 10 ng/L stock concentration of each of forward and reverse primer (Supplemental Table S2 and 3 L of H2O). The reaction was done on Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The qPCR program consisted of 95 C for 5 min, then 40 cycles of 94 C for 15 s, 58 C for 30 s, and 72 C for 30 s and a final melt curve step from 65 to 95 C with a rise of 0.5 C for 5 s. The reactions were performed in duplicates using three biological replicates.