Realstar green fast mixture

For lncRNA and circRNA, total RNA isolation and cDNA preparation from different tissue samples of GRS and HG were the same as those above in “RNA isolation and quality control.” The quantitative real-time PCR (qRT-PCR) analysis was performed using 2 × RealStar Green Fast Mixture (GenStar, China) and the Real-Time PCR System (Lightcycler 96, Roche, Switzerland). β-actin was used as an internal control gene. For each reaction, 0.5 μl of the forward and reverse primers and 2 μl of cDNA template were added.
In PCR validation of miRNA expression, total RNA reverse transcription was performed using the Mir-X miRNA First-Strand Synthesis kit (TaKaRa, Dalian, China). The 5′ forward primers for qRT-PCR validation of miRNAs included the entire sequence of the mature miRNAs, as suggested by the manufacturer, and the 3′primer for qRT-PCR was supplied with the kit. The qRT-PCR analysis was performed using TB Green Premix Ex Taq II (TaKaRa, Dalian, China) and the Real-Time PCR System (Lightcycler 96, Roche, Switzerland). U6 was used as the internal control. For each reaction, 0.8 μl of the forward and reverse primers and 2 μl of cDNA template were added.
All of the primers used in this study are listed in Table S13. The relative gene expression level was calculated according to the 2^−ΔΔCt method⁵⁶ .

Ralstonia solanacearum strain GMI1000 was grown in BG medium at 30°C. The total RNA was extracted from the cells in 0.5 ml of culture from each growth phase with a E.Z.N.A.^® Bacterial RNA Kit (Omega Bio-Tek, Inc.). Once extracted, 1 μg of total RNA was reverse transcribed with the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio Inc.). Quantitative real-time PCR (qPCR) was performed with the 2 × RealStar Green Fast Mixture (GenStar). Relative quantitation was done by the comparative cycle threshold (ΔΔCT) method using the endogenous internal control 16S rDNA and gryB (DNA gyrase subunit B; Castillo and Greenberg, 2007 (link)) for sample normalization. The primers used in reverse transcription PCR (RT-PCR) and qPCR are listed in Supplementary Table 2.

Nodule total RNA was extracted by using RNAiso Plus (TaKaRa). Purified RNA was quantified by using NanoPhotometer Pearl (Implen), and quality was checked by using capillary electrophoresis (Bioanalyzer; Agilent). The resultant RNA samples were subjected to mRNA enrichment, library construction, and strand-specific RNA sequencing (PE150) on an Illumina HiSeq 2000 platform following the manufacturer’s instructions (Illumina) by Novogene (The Genome Analysis Centre, Beijing, China). Two sets of nodules from two independent experiments were used.
qRT-PCR was performed by using primers listed in Table S1. In detail, 8 μg nodule or root RNA was used for reverse transcription. The StarScript II first-strand cDNA kit with genomic DNA (gDNA) remover (Genstar) was used to synthesize cDNA. qRT-PCR was performed by using 2×RealStar Green Fast Mixture (Genstar) and a Roche LightCycler480 II system. Transcription levels were normalized to the expression of the control genes, including 16S rRNA gene of bacteria and an actin gene (Gm.15G05020) of soybean. Triplicates from three independent experiments were used.

qRT-PCR was performed as our previous publication [14 (link), 17 (link)]. Total RNA was isolated from the cells treated with HST or DMSO for 48 h using TRIzol (Life Technologies, Carlsbad, CA, USA). cDNA was synthesized from 1 μg of total RNA using StarScript II First-strand cDNA Synthesis Mix (Genstar, Beijing, China). According to the manufacturer’s instructions, qPCR was further conducted with aliquots of cDNA samples mixed with 2 × RealStar Green Fast Mixture (Genstar, Beijing, China) using a CFX96™ Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA). GAPDH was used as the reference gene for normalization using the 2^−△△Ct method. The sequences of primers were as follows:

p53, Fw 5′-TAACAGTTCCTGCATGGGCGGC-3′,

Re 5′-AGGACAGGCACAAACACGCACA-3′;

p21, Fw 5′-TGTCCGTCAGAACCCATGC-3′,

Re 5′-AAAGTCGAAGTTCCATCGCTC-3′;

MDM2, Fw 5′-AGTAGCAGTGAATCTACAGGGA-3′,

Re 5′-CTGATCCAACCAATCACCTGAAT-3′;

GAPDH, Fw 5′-CATGAGAAGTATGACAACAGCCT-3′,

Re 5′-AGTCCTTCCACGATACCAAAGT-3′;

The RNA was extracted using Quick RNA isolation Kit (Huayueyang Biotech Co., Ltd., Beijing, China). cDNA was synthesized from 1 μg RNA using EasyScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR Kit (TransGen Biotech, Beijing, China). qRT-PCR analysis was performed on a CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA) using 2×RealStar Green Fast Mixture (Genstar, China) with MsTERT gene as reference gene. According to the MsTERT sequence, multiple pairs of primers were designed by primer 5 for pre-experiment. After formula calculation, MsTERT-F/MsTERT-R primers (Table 2) had the highest amplification efficiency (about 95%), so this pair of primers was selected for MsTERT expression pattern analysis in subsequent experiments. Each sample has three technical repetitions. The thermal cycle program was 95 °C for 25 min, 40 cycles of 95 °C for 5 s, and 60 °C for 10 s. The gene expression data are calculated by 2^−∆∆Ct method.

A total of eight differentially expressed P450s from each treatment group were selected for qPCR validation. The relevant primers and internal reference gene (elongation factor-1 alpha, EF1α) information are shown in Table S1. RNA was extracted as described previously. The RevertAid™ Master Mix with DNase I (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to reverse transcribe these into cDNA. Quantitative PCR reactions were performed on a C1000™ Thermal Cycler PCR (Bio-Rad, Hercules, CA, USA) with a reaction volume of 20 μL per sample, including 1 μL cDNA template, 10 μL 2 × RealStar Green Fast Mixture (Genstar, Beijing, China), 1 μL forward and reverse primers, and 8 μL of ddH₂O. Finally, the expression levels of the target genes were calculated according to the method described by Pfaffl [32 (link)].

A total of 15 DEGs with significantly different expression in the TES and PMT were randomly selected for validation of the Illumina sequencing data via a qPCR (LightCycler 480 Roche) analysis. cDNA was synthesized using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO BIOTECH CO., LTD. Shanghai, China) with the total RNA used in the RNA-Seq analysis. The primers designed for amplification were based on RNA-Seq data in our library and NCBI nucleotide databases. All primer sequences are listed in Table 2. The reaction was performed in a 20 μL reaction volume containing 4 μL cDNA, 10 μL 2×RealStar Green Fast Mixture (Genstar, China), 1 μL each primer (10 mM), and 4 μL nuclease-free water. β-actin was used as an internal control to normalize the gene expression level. The PCR conditions were as follows: 10 min at 95 °C, 40 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and followed by a cooling step at 4 °C. Every sample was amplified in triplicate to normalize the system and reduce pipetting errors. The 2^−ΔΔCT method was used to analyze the relative expression level. Pearson’s correlation coefficient was used to assess the expression data consistency between the RNA-Seq and qPCR.