Hiscript 2 1st strand cdna synthesis kit

To identify the possible causes of illness, the serum samples from cattle with clinical symptoms were pooled for viral metagenomic analyses as previously described [19 (link)]. To further validate the presence of GETV, viral RNA was extracted using a QIAamp Viral RNA Mini Kit (Qiagen, USA). The RNA was converted into cDNA using a Vazyme HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., China) in accordance with the manufacturer’s instructions. RT-qPCR was performed with RNA from the cattle serum and mosquito samples as described elsewhere [20 (link)]. Serum neutralization (SN) tests were carried out using the microtiter method as previously described [21 (link)].

Walnut female bud samples fixed in FAA for 24 h and softened by glycerol immersion were treated with a continuous gradient of ethanol (70, 85, 95, and 100%) and a mixture of anhydrous ethanol and xylene (v:v = 1:1) for dehydration, and then were immersed in 60°C liquid paraffin. Slicing was done using a Leica Microtome (Leica, Weztlar, Germany) with a thickness of 8 μM, sections were stained using Fast Green FCF (Sangon Biotech, Shanghai, China), Nikon Eclipse Ts2 microscope (Nikon, Shanghai, China) was used for observation of the sections, and images were processed using NIS-Elements.
Total RNA was extracted from different tissues by RNA extraction kits (Tiangen, DP441, Beijing, China). First-strand cDNA was synthesized using Vazyme HiScript II 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., China). Specific primers were designed based on 18 selected coding sequences of JrASMTs (Supplementary Table 1), and Sangon Biotech Co., Ltd. (Shanghai, China) synthesized the primers. The Actin gene was used as an internal reference and real-time fluorescence quantification by Maxima SYBR Green/ROX qPCR Master Mix. Three biological replicates were included in this experiment, and gene expression was calculated by 2^−ΔΔCt (Livak and Schmittgen, 2001 (link)). qP1 period samples were set as the control.

SLK.iBAC-GFP or BCBL1-Tet-K-RTA cells were treated with Dox (1 μg/ml) for 24 h. Total RNA was extracted using TRIzol reagent (Takara) and cDNA was synthesized using HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Then the cDNA mixture was diluted 40-times and subjected to qPCR analysis with SYBR green qPCR master mix (Bimake, Shanghai, China). The relative quantitation of the target genes was normalized to ATCB. The primer sequences are listed in S2 Table.