Cfx96tm system

In the validation experiments based on qRT-PCR analysis, a total of 12 genes were randomly selected to assess the reliability of RNA-Seq data (Supplementary Table 3). The RNA sample of 1 μg was reverse-transcribed into cDNA using the PrimeScript™ RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer's instructions. The cDNA was diluted 10-fold and used for qRT-PCR analyses with a Bio-Rad CFX96TM System and signal detection protocols in accordance with the manufacturer's instructions (TaKaRa, Dalian, China). The qRT-PCR experiments were performed in three replicates with the β-Actin used as the endogenous control and the expression of individual genes normalized to that of β-Actin (Xiang et al., 2017 (link)). Primers used for qRT-PCR experiments are shown in Supplementary Table 3.

Total RNA was extracted from cell samples, using a RNeasy Mini Kit (Cat#74104, Qiagen), according to the manufacturer's instructions. The concentration and quality of the extracted RNA were measured using a NanoDrop2000 Spectrophotometer (Thermo Scientific). 5 μg of RNA from each sample was transcribed into cDNA using Tetro cDNA Synthesis Kit (Bioline) following manufacturer's instructions. Primers used for gene expression analysis, listed in Table 1, have been published previously [32 (link)].
qRT-PCR was performed by using SsoAdvancedTM Universal SYBR Green Supermix kit (ThermoFisher Scientific). Three independent experiments were performed in triplicate, with GAPDH as the reference gene. The reaction mix preparation and thermal cycling protocol were followed according to SsoAdvancedTM Universal SYBR Green Supermix kit. A Bio-Rad CFX96TM system was used for thermal cycling, with initial denaturing at 95°C for 30 sec, then 40 cycles of denaturing at 95°C for 10 sec, annealing and extension at 59°C for 30 sec, and Melt-Curve Analysis from 65°C to 95°C with 0.5°C increment. Statistical analysis was performed by one-way ANOVA; the P5H samples were set to a value of 1 and used as a reference to determine a statistical significance.

The protocol was followed by the established method [31 (link)]. RAW264.7 cells were cultured with PIMP in the presence or absence of LPS (1 μg/mL). Using the TRIzol reagent, total RNA was extracted from the colon tissue and RAW264.7 cells in accordance with the guidelines provided in the manual. Using a Transcription cDNA Synthesis Kit, the extracted RNA was reverse-transcribed into complementary DNA (cDNA). Table S1 contains primer information. The CFX96^TM system (Bio-Rad, Hercules, CA, USA) in conjunction with the FastStart SYBR Green Master Mix (Takara SYBR^® Pre-mix Ex Taq^TM II, Dalian, China) was used to conduct the PCR reactions. A typical two-step protocol was used for the PCR amplification: 40 cycles of 95 °C for 3 s and 60 °C for 30 s were followed by one cycle at 95 °C for 30 s. The samples were subjected to melting curve analysis. The internal reference for relative expression was GAPDH mRNA.

Total RNA was isolated from the leaf samples (100 mg) using TRIZOL reagent (GIBCO-BRL) as per instructions given in the manufacturers’ protocol. The total RNA was digested with DNase at 37 °C for 15 min and then reverse transcribed into cDNA using M-MLV Reverse Transcriptase RNaseH (Bio-Rad CFX-96^TM system). Primers were designed using the software Primer 3 (CHS1 forward: 5′-AGCCAGTGAAGCAGGTAGCC-3′, CHS1 reverse: 5′-GTGATCCGGAAGTAGTAAT-3′ and CHS2 forward: 5′-AGCCAGTGAAGCAGGTAGCC-3′, CHS2 reverse: 5′- GTGATCCGGAAGTAGTAAT -3′), referring to accessed sequences in the Genbank (Supplementary Table S5). The semi quantitative RT-PCR of selected genes was done according to Goto-Yamamoto et al. (2002)^{61 (link)}. Triplet of all sample reactions were carried out and negative control of master mix in addition to primers was performed in all RT-PCR runs. GAPDH was taken as control because of its constitutive expression. The accuracy of primers was tested using genomic DNA of the plant as positive control. The intensities of the PCR products on agarose gels were quantified with the Gel Doc 2000 system and volume tool of the Quantity one software (BioRad, USA).

Total RNA was extracted for mRNA blot analysis and cDNA synthesis as previously described [29 (link)]. Quantitative real-time PCR (qRT-PCR) was carried out in triplicate using the primers listed in Table S3. Real-time RT-PCR was performed on a BioRad CFX96TM system using 25-µL mixtures containing 10 ng of synthesized cDNA, 1× iQ SYBR Green Supermix (BioRad, Hercules, CA, USA), and 0.2 µM forward and reverse primers. Relative expression levels were calculated on the basis of serial dilutions of cDNA (125–0.2 ng), which were used to generate standard curves for each gene. Cycling conditions consisted of a single incubation step at 95 °C for 5 min followed by 30 cycles of 95 °C for 10 s, 58 °C for 35 s, and 72 °C for 15 s. Specificity was confirmed by product melt curve analysis over the temperature range 50–90 °C with fluorescence acquired after every 0.5 °C increase, and the fluorescence threshold value and gene expression data were calculated with BioRad CFX manager software version 3.1. Values represent the mean of three biological replicates ± SE. Amplification efficiencies were compared by plotting the ΔC_t values of different primer combinations of serial dilutions against the log of starting template concentrations using the CFX96TM software. Differences among samples were observed by mean ± SE.

The real-time PCR assay was performed thrice for each sample using the SYBR^® Premix ExTaq^TM kit (TaKaRa). Each PCR amplification procedure was performed with the CFX96^TM system (Bio-Rad) at a total volume of 25 μl, following the manufacturer’s instructions. The U. maydis Pep1-specific primer set of Pep-qF (5′-ACAATTCGTACACACTGCCG-3′) and Pep-qR (5′-CCCTTCTTCTCCTGGTCGTT-3′) was used, which was designed with the Primer3 web version 4.0 (http://primer3.ut.ee/)^{43 (link)}. The thermal cycling conditions were set up as previously described^{27 (link)}.

RNAprep Pure Plant Kit (Polysaccharides&Ployphenolics-rich, DP441) was used to extract RNA from four different stages of fruit (Tiangen Biotech, Beijing, China). Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 22 March 2022) was used to create the primers utilized in the experiment (Table S5). HiScript^® II 1st Strand cDNA Synthesis Kit (+gDNA wiper) was used to make the first-strand cDNA (Vazyme, Nanjing, China). The internal reference gene is malate dehydrogenase (MDH) [74 (link)]. On a Bio-Rad CFX96TM system, real-time quantitative PCR (RT-qPCR) analysis was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China).