Nanodrop 2000 instrument

Total RNA was extracted from tissue and cells (1×10⁶) with TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The quantity and concentration of total RNA were determined by a NanoDrop 2000 instrument (Thermo Fisher Scientific, Inc.). RT-qPCR was performed as described previously (33 (link)). Briefly, total RNA was reverse transcribed to cDNA using a PrimeScript RT Reagent kit (Takara Biotechnology Co., Ltd.). qPCR was performed in a 96-well plate on an ABI 7500 system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with PowerUp SYBR-Green Master Mix (Thermo Fisher Scientific, Inc.) as per the procedure provided by the manufacturer. For detecting hsa-miR-23a-5p, a hsa-miR-23a-5p-specific stem-loop primer (Guangzhou RiboBio Co., Ltd.) was used for reverse transcription and RT-qPCR amplification was performed using the Bulge-Loop miRNA RT-qPCR Starter kit (Guangzhou RiboBio Co., Ltd.). The thermocycling conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 60 sec. GAPDH (for circRNA and mRNA) or U6 (for miRNA) was used as reference control. Relative expression level was calculated by the 2^−ΔΔCq method (34 (link)). Primer sequences are listed in Table II.

To analyze nitrate-induced gene expression, M. truncatula wild-type (R108), cra2, nin-2, or nlp1 seedlings were grown on BNM or FP plates supplemented with 50 nM aminoethoxyvinylglycine (Sigma-Aldrich) for 3 days, then transferred to plates with BNM or FP containing 10 mM KCl or KNO₃ for another 3 days. To analyze rhizobium-induced gene expression, R108, nin-2, or nlp1 seedlings were grown on BNM or FP agar plates for 3 days, then inoculated with Sm1021 (OD₆₀₀ ≈ 0.05) and grown for another 3 days. Total RNA was extracted from M. truncatula roots using the TRIpure isolation reagent (Aidlab, Beijing, China) according to the manufacturer's instructions. RNA was quantified using a NanoDrop 2000 instrument (Thermo Fisher, Waltham, MA, USA) and then reverse transcribed into cDNA using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). Gene transcript levels were analyzed by real-time PCR using TB Green Premix Ex Taq (Takara, Dalian, China) with the StepOnePlus PCR system (Thermo Fisher, Waltham, MA, USA). Relative transcript levels were normalized against that of the reference gene (MtUBQ10). Statistical significance based on three biological replicates was calculated using the 2^−ΔΔCt method. The primers used for qRT-PCR are listed in Supplemental Table 1.

Total RNA was extracted from the cells using RNAiso Plus reagent (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. The purity and concentration of RNA was measured using a NanoDrop 2000 instrument (Thermo Fisher Scientific, Inc.). The RNA was transcribed into cDNA using a microRNA Stem-Loop Reverse Transcription kit (GenePharma, Shanghai, China), according to the manufacturer's protocol. To evaluate c-FOS and HMGA2 expression, corresponding RNA was reverse-transcribed into cDNA using the PrimeScript™ RT Reagent kit with genomic DNA Eraser (Takara Bio, Inc.) 48 h after transfection in A549 cells. qPCR was performed with a TransStart Top Green qPCR SuperMix kit (TransGene Biotech Co., Ltd, Beijing, China) using the StepOne Plus system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling settings were as follows: 30 sec at 94°C, then 40 cycles of 5 sec at 94°C and 30 sec at 60°C. Small nuclear RNA(U6) was used as an internal marker of miRNA. For the analysis of c-FOS and HMGA2 expression, β-actin was used for normalization. All reactions were performed in triplicate. The 2^−∆∆Cq method was used for relative quantification of gene expression (17 (link)). The primers used are listed in Table I.