Rnaiso plus extraction reagent

Total RNA from each sample was isolated using RNAiso Plus extraction reagent (TaKaRa, Shiga, Japan). Total RNA integrity was evaluated using 1.5% agarose electrophoresis, and the concentration was quantified by a spectrophotometry (SimpliNano, GE, United States). Residual genomic DNA was removed using DNase I (Thermo Scientific, United States), and 1 μg of RNA was reverse-transcribed into first-strand cDNA with an oligo (dT)18 primer using Revertaid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, United States). The products were stored at -80°C.

Total RNA was isolated from the functional leaf, peduncle, and outer glume of individual plants of the two wheat cultivars grown under various N levels, using RNAiso Plus extraction reagent (Takara, Tokyo, Japan). The RNA quality was confirmed with a 1% agarose gel and quantified with a spectrophotometer. Three replicates were carried out for RNA isolation. Total RNA from each replicate (2 μg) and Hi-Script Reverse Transcriptase with gDNA Wiper Mix (Vazyme Biotech, Nanjing, China) was used to prepare cDNA. The conditions for a 20 μL-reaction were as follows: 2 μL total RNA, 5 μL 4 × gDNA Wiper Mix, and 9 μL RNase-free H₂O at 42 °C for 2 min to digest the DNA, followed by the addition of 4 μL 5 × SuperMixII and incubation at 25 °C for 10 min, 42 °C for 30 min, with a final denaturation step at 85 °C for 5 min.

C. cinerea mycelia in the fermentation broth samples were collected by filtration, and then their total RNA and corresponding cDNA were obtained using RNAiso-Plus extraction reagent (TaKaRa, Dalian, China) and Evo M-MLV RT kit (AG, Hunan, China), respectively. The qualities of the cDNA samples were measured using a Qubit 4.0 fluorometer and Qubit dsDNA HS Assay Kit (Invitrogen, USA). To normalize the qRT-PCR data, the β-actin gene was chosen as the reference based on our previous study (Liu et al. 2022 (link)). Primers used for qPCR amplification of the chitinase and β-actin genes are shown in the Supplementary information file 1: Table S1. qPCR was performed based on the Light-Cycler 96 Real-Time PCR system (Roche, Basel, Switzerland) using the SYBR Green Premix Pro Taq HS qPCR Kit (AG, Hunan, China). The qPCR amplification conditions included 95 ℃ for 30 s, followed by 35 cycles of 95 ℃ for 5 s, 60 ℃ for 30 s, and a melt-curve step (0.3 ℃/s, from 60 to 95 ℃). Real-time PCR data were calculated using a 2^−ΔΔCT methodology (Livak and Schmittgen 2001 (link)). The transcript levels of each gene are presented separately as their fold change relative to that of the internal reference gene β-actin.

Total RNA from human peripheral blood monocytes was extracted with RNAiso Plus extraction reagent following the manufacturer's instructions (No. 9108, Takara, Dalian, China). Total RNA was quantified on a NanoDrop ND-2000 (Thermo Fisher Scientific, USA). For mRNA expression analysis, cDNA was synthesized with reverse transcriptase (TaKaRa Biotechnology, Otsu, Japan) and real-time PCR was performed with TB Green® Premix Ex Taq™ II (RR820L, Takara, Dalian, China) through an ABI-7500 Real-Time PCR Detection System (Applied Biosystems, USA). GAPDH was used as an internal control. The primer sequences used for qPCR are listed in the Supplementary Material. The relative gene expression level was calculated with the comparative 2 ^-∆∆CT method and normalized to GAPDH expression.

Total EV-associated RNA was extracted with RNAiso Plus extraction reagent following the manufacturer's instructions (Cat. 9108, Takara, Dalian, China). Stem-loop primers (purchased from Ribobio Biotech) were used to produce cDNA from miRNA using a PrimeScript RT reagent Kit (Cat. RR047A, Takara). The miRNA cDNA was amplified using TB Green® Premix Ex Taq™ II (Cat. RR820L, Takara) on an ABI-7500 Real-Time PCR Detection System (Applied Biosystems, USA). U6 snRNA was used as internal control.