Rna easy fast plant tissue kit

Approximately 0.1 g frozen leaves of upland cotton were crushed into powder in liquid nitrogen. The supernatant were collected using centrifugation and transferred to a new centrifuge tube. Then, we used the RNA Easy Fast Plant Tissue Kit to extract total RNA (Lysis Buffer, Proteinase K and RNA Easy Fast Plant Tissue Kit were brought from TIANGEN BIOTECH Co., Ltd., Beijing, China) (Liu et al., 2019 (link)). Total RNA was reversed into cDNA using the PrimeScript^TM RT reagent Kit with gDNA Eraser (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). The upland cotton gene GhUBQ7 worked as an internal reference. Quantitative analysis was performed using AceQ^® qPCR SYBR Green Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China) and a real-time qPCR system (ABI Step One Plus™), with three biological repeats (Li et al., 2017 (link); Wu et al., 2017 (link)).

To validate the salt-responsive m6A genes, 5 candidates obtained by RNA-seq analysis were randomly selected to investigate their expression patterns under salt stress using qRT-PCR method. Seeds of wild emmer wheat genotype As2389 were hydroponic cultured in the growth chamber under controlled conditions (22 ± 1 °C, 16 h light/8 h dark cycle). The three-week-old seedlings were subjected to 200 mM NaCl solution, and samples were collected after 0 h, 3 h, 6 h, 12h and 24 h treatment, respectively. Leaves of all samples were collected from three to five plants at each time point with three biological replications. All samples were stored at −80 °C for RNA extraction using RNA Easy Fast Plant Tissue Kit (Tiangen, Beijing, China). cDNA was synthesized using RT Master Mix Perfect Real-Time kit (Takara, Dalian, China), and qRT-PCR reactions were performed using SYBR^® Green Premix Pro Taq HS qPCR Kit (Ac-curate Biology, Changsha, China) according to the manufacturer’s protocol. qPCR was performed on the QuantStudioTM 7 Flex System (Thermo Fisher Scientific, Waltham, MA, USA) with the thermal cycling condition was 95 °C for 30 s followed by 40 cycles of 95 °C for 5 s, 58 °C for 34 s. TaELF-1 was used as the internal reference, and the primers used in this study were listed in Table S8. The relative expression levels were determined by 2^−ΔΔCt method.

CGMMV-infected watermelon leaves showing typical symptoms of chlorotic mottling were collected from Sanya City in Hainan Province, China, in the growing season of 2020. Nontarget viral isolates, including Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Melon mosaic virus (MMV), Tobacco ring spot virus (TRSV), Cucurbit chlorotic yellows virus (CCYV), Zucchini yellow mosaic virus (ZYMV), Squash mosaic virus (SqMV), Melon yellow spot virus (MYSV), Melon necrotic spot virus (MNSV), Prunus necrotic ringspot virus (PNRSV), Papaya ringspot virus (PRSV), Tobacco mosaic virus (TMV) and Potato Y virus (PVY), were provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences (Table 1). All of the virus isolates used in this study were detected and confirmed previously by RT-PCR and stored at −80 °C.
Total RNA was extracted by using an RNA Easy Fast Plant Tissue Kit (TianGen Biotech Co., Ltd., Beijing, China) following the manufacturer’s instructions. Then, 1% agar gel electrophoresis was performed to determine RNA integrity. RNA concentration and purity were measured by using a SmartSpec Plus spectrophotometer (Bio-Rad Laboratories Inc., Hercules, CA, USA). RNA samples with A260/A280 ratios within the range of 1.9–2.1 were used as templates for RT-qPCR. The RNA samples were stored at −80 °C.