Nanodrop 2000c uv vis spectrophotometer

A total of 100 mg of fresh leaves was collected from each of the 1882 lettuce samples (71 parents, 871 progeny and 940 F3) and ground to a fine powder using Tissue Lyser II (Qiagen, Valencia, CA, USA). Genomic DNA (gDNA) was extracted with the Dneasy^® 96 Plant Kit (Qiagen), according to the manufacturer’s protocols. After extraction, the integrity of the gDNA was assessed by electrophoresis on 1% (w/v) agarose gel stained with SYBR Safe^® 1 × DNA Gel Stain (Life Technologies, Carlsbad, CA, USA) in Tris-Acetate-EDTA (TAE) running buffer. Both the yield and purity of the extracted gDNA samples were evaluated using a NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Scientific, Carlsbad, CA, USA). Following DNA quantification, all DNA samples were diluted to a final concentration of 20 ng/μL.

Mature rice seeds were ground to a fine powder and extracted overnight at 4 °C in five volumes of lysis buffer (50 mm NaH₂PO₄, 300 mm NaCl, 5 mm imidazole, pH 8). Insoluble material was removed by centrifuging twice at 8000 g for 30 min at 4 °C. Each sample was loaded onto a Profinity IMAC column at a flow rate of 2 mL/min. The column was washed twice with wash buffer (50 mm NaH₂PO₄, 300 mm NaCl, 10 mm imidazole, pH 8) and the protein was eluted four times with elution buffer (50 mm NaH₂PO₄, 300 mm NaCl, 250 mm imidazole, pH 8). Protein‐containing fractions were identified using a NanoDrop 2000c UV‐Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA) based on extinction coefficient at 280 nm of the protein, and GRFT concentrations were determined by ELISA (see above). Fractions containing >50 μg/mL GRFT were pooled and concentrated by ultrafiltration using spin columns with a 3 kDa molecular weight cut‐off (Amicon Ultra, EMD Millipore, Darmstadt, Germany).

Genomic DNA was isolated from blood samples using a QIAamp DNA Blood Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Genotyping for TLR9 (−1237T/C, rs5743836; −1486T/C, rs187084; 1174G/A, rs352139; and 2848C/T, rs352140) SNPs were performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) as we described elsewhere [10 (link)]. The reaction was performed in a Veriti 96 Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The concentration and purity of DNA were determined using a NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The digested fragments were analyzed using a QIAxcel system (Qiagen). The selected samples of each TLR9 SNP were sequenced using the 96-capillary 3730xl DNA Analyzer (Applied Biosystems) to confirm the detected genotypes.

Total RNA was extracted from gonad in different development stage of rice field eel (n = 4) using Total RNA kit II (Omega bio-tek) according to manufacturer's instructions. Total RNA concentrations were measured at 260 and 280 nm using a Nanodrop 2000c UV-Vis spectrophotometer (Thermo Scientific, Waltham, Mass., USA). A 260 nm reading was used to determine the concentration of total RNA and the quality was verified by measuring the 260/280 and 260/230 ratios. Total RNA (1.5 μg) was reverse-transcribed to cDNA using a HiScript II Q RT SuperMix for qPCR with gDNA wiper kit (Vazyme). Primer sequences for cloning and qPCR were designed and purchased from Integrated DNA Technologies (Tianyi Huiyuan, Wuhan, China) and PCR product sizes are listed in S1 Table.
Quantitative real-time RT–PCR experiments were performed in a final volume of 20μL containing 1μL of original cDNA, 0.8μL of each 10 mM primer, 0.4 μL 50× Rox reference, 7.8 μL of RNase and DNase free H₂O, and 10μL of SYBR Green Master Mix (AceQ qPCR; Vazyme). The protocol was: 7 min at 95°C, followed by 40 cycles of 95°C for 10s, 60°C for 30s, and 72°C for 30s. qPCR was performed in triplicate using Rotor-Gene Q (Qiagen). A melting curve was generated for each run to confirm the specificity of the each product. Fold change was calculated by normalized Ct values against rpl17 [38 (link)] gene using the 2^-ΔΔct method[39 (link)].

CCMV was harvested and purified from infected Cowpea plants (Vigna ungiculata) following established protocols [4 (link),50 (link)]. Briefly, it was dialyzed against disassembly buffer (500 mM CaCl₂, 50 mM Tris-HCl pH = 7.5, 1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF) for 24 h in order to disrupt the viral capsids and separate the CP and the RNA. Then the disassembled virus was ultracentrifuged, at 536,600 g using a TLA110 rotor for 106 min at 4 °C, in order to precipitate the RNA and recover the purified protein still in suspension. The recovered protein was then dialyzed in protein buffer (1 M NaCl, 50 mM Tris-HCl pH = 7.2, 1 mM EDTA, 1 mM DTT, 1 Mm PMSF) that stabilized the protein into dimers [51 (link)]. Protein concentration and purity was measured in a UV-Vis spectrophotometer (NanoDrop 2000c UV-Vis Spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA), and only the fractions with 280/260 ratios above 1.5 were chosen and used for the encapsidation experiments.

The collected plant samples were ground to a fine powder in liquid nitrogen. Total RNA was extracted from the ground material with TRIzol reagent (Takara, Dalian, China). Specifically, the powdered samples were resuspended in TRIzol reagent and then mixed by vortexing. Samples were centrifuged at 12,000×g for 5 min at 4 °C. Phenol–chloroform equal to one-fifth of the total volume was added to the supernatant and the resulting solution was centrifuged at 12,000×g for 5 min at 4 °C. An equal volume of chloroform was added and the solution was centrifuged at 12,000×g for 5 min at 4 °C, after which a half volume of 8 M LiCl and a half volume of 75% alcohol were added to the supernatant to precipitate the RNA for at least 1 h. Finally, 75% ethanol was used to purify the RNA, which was finally dissolved in RNAase-free water (Zhang et al. 2018 (link)). The RNA concentration was determined with the NanoDrop 2000c UV–Vis spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
The total RNA was used as the template for the synthesis of cDNA with the HiScript II Q Select RT SuperMix reagent Kit (Vazyme Biotech Co., Ltd, Nanjing, China). The cDNA templates were diluted 1:5 with nuclease-free water and stored at − 20 °C until analyzed in a quantitative real-time polymerase chain reaction (qRT-PCR) assay with minimal thawing and refreezing.