Tripure reagent

Ten days old A. thaliana seedlings grown at 22 °C and TriPure Reagent (Aidlab Biotechnologies Co., Ltd., Beijing, China) were used for the isolation of total RNA. Real-time quantitative RT–PCR (Q–PCR) was conducted following the method of Zhang et al. [53 (link)]. Primer Express software (Applied Biosystems, Carlsbad, CA, USA) was used to design Q-PCR primers (Table S1). PCR was conducted using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Carlsbad, CA, USA). The expression levels of genes tested in the WT or control plants were set to 1. ACTIN1 gene was used as the internal control.

The RAW 264.7 macrophages were co-treated with ZQE (250 μg/mL) or aspirin (250 μg/mL) and LPS (1 μg/mL) for 12 h, while the primary BMDCs with ZQE (1 μg/mL) and LPS (100 ng/mL) for 8 h, respectively, to measure the mRNA expression of COX-2, IL-1β, IL-6, and TNF-α. Total RNA was isolated from these cell samples according to the manufacturer’s instruction of TRIpure reagent (Aidlab, Beijing, China), and then the first-strand copy DNA (cDNA) was synthesized using a reverse transcription kit (Takara, Japan). Quantitative real-time polymerase chain reaction (qPCR) was performed in an ABI 7500 Real-Time System with SYBR Green PCR Master Mix (Takara). Reactions were initiated with an initial incubation at 50 °C for two minutes and 94 °C for 10 min, followed by 40 cycles of 94 °C for 5 s, 60 °C for 15 s, and 72 °C for 10 s. The relative gene expression levels were calculated using the 2^−ΔΔCt method. The gene primers for qPCR are provided in Table 3. β-actin was used as an internal reference gene between different samples.

For quantitative real-time PCR analysis, samples were prepared from leaves harvested 6 days after heat stress. The samples were frozen in liquid nitrogen and stored at −80 °C. Total RNA was extracted from the samples using TRIpure reagent (Aidlab Biotechnologies, Beijing, China). RNA was converted into first-strand cDNA using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Shanghai, China) and oligo (dT) as the primer. The resultant cDNA was used as the template for quantitative PCR amplification in a Thermal Cycler Dice Real-Time System II (TaKaRa Biotechnology, Dalian, China) with SYBR Green I (TOYOBO) as the fluorescent reporter. Primers were designed to generate 150–250 base pair (bp) fragments using the PRIMER3 software [37 (link)] (Table S3). PCR analysis and detection were performed as described previously [38 (link)]. The 2^−ΔΔCT method was employed to analyze the relative gene expression levels using the mean values from three replicates.

The RAW 264.7 macrophages were cultured according to a previously published method (Cheng et al., 2017). Briefly, the RAW 264.7 cells were grown in 25 cm² cell culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml). The cells were incubated at 37°C and 5% CO₂‐95% air in a humidified incubator. Near confluent cells were removed with a cell scraper, and then seeded on cell culture plates for further experiments.
To evaluate the immune activity of WSS under different storage conditions, the cells were treated with different WSS samples (0.5 mg/ml). After incubation for 18 hr, total RNA was isolated from the cells with TRIpure reagent (Aidlab, Beijing, China) according to the manufacturer's instructions. And the cDNA was synthesized using a reverse transcription kit (Vazyme). The relative mRNA expression levels of proinflammatory cytokines were detected by using an ABI QuantStudio 6 Flex Real‐time PCR System with SYBR Green PCR Master Mix (Yeasen). Reactions were cycled as follows: 50°C for 2 min, 95°C for 5 min; 95°C for 10 s, 60°C for 30 s (40 cycles). And the primer sequences of proinflammatory factors including cyclooxygenase‐2 (COX‐2), inducible nitric oxide synthase (iNOS), interleukin‐6 (IL‐6), and interleukin‐1β (IL‐1β) are referred to Cheng et al. (2017).

Total RNA was extracted using TRIpure reagent (Aidlab, Beijing, China). The cDNA was synthesized using HiScript^® 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The target gene expressions were examined using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA, USA). The qPCR program was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 1 min. β-actin served as an internal reference control. The relative mRNA levels were calculated using the 2^−ΔΔCT method. For all the qPCR assays, an efficiency comprised between 90 and 110% was measured. The sequence of qPCR primers used in this study are listed in Table S1.

To analyze the infection rate of PKV and PEDV in Chinese swine herds in recent years, total RNAs were extracted from these clinically intestinal samples using TRIpure Reagent (Aidlab, Beijing, China) according to the manufacturer’s instructions. cDNAs were produced via reverse transcription using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper, Vazyme, Nanjing, China). The obtained sample cDNAs were tested using our previously described PKV and PEDV real-time RT-PCR (qPCR) assays [32 (link)] using StepOne Plus Real-time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) and Q711-00 hamQ Universal SYBR qPCR Master Mix (Vazyme, China). In addition, all PKV qPCR amplicons were further confirmed through Sanger sequencing (Genewiz, Suzhou, China). Three PKV-positive samples from three cities of three provinces (Yangzhou city of Jiangsu province, Kashi city of Xinjiang province, Chaozhou city of Guangdong province) and from the period of 2018 to 2022 were selected as representative samples and submitted for genome sequencing (Table 1). The PKV genome amplification of a representative sample (GDCZ2202-1606) containing seven overlapping fragments is shown in Figure 1.

Roots, stems, leaves, ovaries, prickles, and stamens were sampled for TCP genes expression analysis via qPCR. All samples were immediately frozen in liquid nitrogen for two hours and stored at −80 °C before use. Total RNA was extracted using TRIPure reagent (Aidlab Biotechnologies, Beijing, China). First-strand cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Shanghai, China). The Step One Plus^TM Real-Time PCR Instrument Thermal Cycling Block (Thermo Fisher Scientific Biotechnology, Shanghai, China) was used for qPCR analysis. Eight genes were selected to study the sequencing data. The primes are listed in Supplementary Table S2. The thermos cycle was set as follows: 95 °C for 30 s; 40 cycles of 95 °C for 10 s; 60 °C for 30 s. RcUbi was used as an internal control. The 2^−∆∆CT method was employed to analyze relative gene expression levels using the mean values of three replicates.