Primescript rt reagent kit with a gdna eraser

The RNA preparation and RT-qPCR protocols were as described previously [11 (link)]. Briefly, total RNA was extracted from all the samples with Trizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration of the extracted RNA was measured in a NanoDrop ND-1000 spectrophotometer (Agilent Technologies, Palo Alto, CA, USA). The purity of the RNA was measured based on the A260:A230 and A260:A280 ratios, and the quality of RNA was analyzed by 2% agarose gel electrophoresis.
The A260:A230 and A260:A280 ratios ranged from 2.00 to 2.10 and 1.90 to 2.05, respectively, suggesting that all the RNA samples were of high quality. To synthesize the cDNA, 1.00 μg of total RNA from each sample was reverse transcribed using a PrimeScript™ RT Reagent Kit with a gDNA Eraser (Takara Bio Inc., Otsu, Japan), according to the manufacturer’s protocol.

The qRT-PCR was performed as previously described (Gu et al., 2020 (link)). The WT, ΔrpoN, ΔrpoNΔopaR, ΔrpoNΔqrr2, and ΔrpoNopaR⁺ strains were cultured at 37°C in LB medium overnight and diluted 1:100 in new LB medium for 5–6 h or in the BHI agar for 48 h. Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Genomic DNA was removed using RNase-free DNase I. Equal amounts of RNA (1 μg) were used to generate the first-strand cDNA using the PrimeScript RT Reagent Kit with a gDNA eraser (Takara, Tokyo, Japan). The specific primers used for qRT-PCR are listed in Supplementary Table S2. The reactions were performed on the ABI PRISM 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) with a FastStart Universal SYBR Green Master (Roche, Mannheim, Germany). The transcript levels of each sample were normalized to those of gyrB using the 2^−ΔΔCt method. Three independent experiments were performed, and each experiment was run in triplicate.

At the corresponding time point, 5 mL of the bacterial solution was collected in a sterile 15 mL centrifuge tube and centrifuged at 8000 rpm for 20 min at 4 °C to obtain the bacterial precipitate. Subsequently, 1 mL of sterile PBS solution was added to the centrifuge tube, and the bacterial precipitate was resuspended using a pipette gun. The resuspended bacteria were then transferred to a new sterile 2 mL centrifuge tube and re-centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was discarded, and the process was repeated once. After aspirating the solution in the centrifuge tube, 100 μL of lysozyme solution (3 mg/mL) was added to resuspend the bacteria by gently blowing with a pipette before bathing in water at 37 °C for 10 min.
Total RNA extraction from bacteria was performed using the TIANGEN RNAprep Pure Cell/Bacteria Kit (TIANGEN BIOTECH (Beijing) Co., Ltd., Beijing, China), followed by reverse transcription of the tRNA to cDNA using the PrimeScript RT Reagent Kit with a gDNA Eraser (Takara Biotechnology (Dalian) Co., Ltd., Dalian, China). Finally, the quantitative real-time PCR assay was performed on the Roche LightCycler96 PCR detection system (Roche, Switzerland), according to optimized PCR protocols, using the TB Green Fast qPCR Mix (Takara Biotechnology (Dalian) Co., Ltd.). The primer pairs were used as listed in Table S1.

Total RNA was extracted using the E.Z.N.A. Total RNA kit I (Omega Bio-tek Inc, Norcross, GA, USA). Next, mRNA was reverse transcribed and synthesised into complementary DNA using the PrimeScript™ RT Reagent Kit with a gDNA Eraser (RR047A, Takara, Japan) following the manufacturer’s protocol. Real-time RT-PCR was performed in triplicate using FastStart Essential DNA Green Master (Roche, Penzberg, Germany). Primer sequences are listed in Additional file 1: Table S1. All results are presented as fold differences normalised to GAPDH. The data were analysed by the comparative threshold cycle method defined as 2^−ΔΔCT.

Total RNA was isolated from rice leaf samples (100 mg tissue per sample) using Trizol reagent (Invitrogen, Gaithersburg, MD, USA). Concentration of total RNA in each sample was determined using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized using one μg total RNA per 20 μL reaction using the PrimeScript™ RT reagent Kit with a gDNA Eraser (Takara, Dalian, China). qPCR was then performed using the SsoFast EvaGreen® Supermix (Bio-Rad) on a Bio-Rad iQ5 qRT-PCR system. The expression levels of OsUBC and OsActin1 were determined and used as internal controls as previously reported (Fang et al. 2015 (link); Lu et al. 2016 (link)). qPCR primers specific for RBSDV P10, OsNOA1, OsNIA2, OsPR1b, OsWRKY45 or OsICS1 are listed in the Supplementary Table 1. The qRT-PCR results were calculated using the 2^-ΔΔCt method reported previously (Livak and Schmittgen 2001 (link)).

The total RNA was isolated from 8-day-old A. suturalis females using the TRIzol reagent (Invitrogen, LifeTechnologies, Carlsbad, CA, USA). The total RNA (1 μg) was reverse transcribed using a PrimeScript™ RT Reagent Kit with a gDNA Eraser (Takara, Kyoto, Japan) and was then employed as a template to amplify fragments of AsAP, according to A. suturalis transcriptome data [21 (link)]. A smart kit for the rapid amplification of cDNA ends (RACE) (Takara, Kyoto, Japan) was used to facilitate the amplification of full-length AsAP, according to the manufacturer’s instructions. The resulting product was gel-purified (AxyGen, Suzhou, China), ligated into a pEASY-T1 vector (TransGen, Beijing, China) and sequenced. The resultant sequence information was submitted to the National Center for Biotechnology Information (accession number: OP019357). All primers that were used are indicated in Table 1.
The AsAP amino acid sequence was deduced using the ExPASy Translate tool, the putative functional domains were predicted using the EMBL SMART program [22 (link)], the theoretical molecular weights were determined using PeptideMass [23 (link)] and the putative asparagine-linked glycosylation sites were determined using NetNGlyc 1.0 [2 (link)]. Our phylogenetic analyses were conducted based on the neighbor-joining method and a Jones–Taylor–Thornton substitution in MEGA 7.0 [24 (link)].