Cdna synthesis supermix

Total RNA was isolated from leaves using the TRIZOL reagent following the procedure of the manufacturer. The first strand of cDNA was synthesized with 1.0 μg RNA using cDNA Synthesis SuperMix (TransGen). The real-time quantitative PCR (RT-qPCR) analysis was performed on a StepOne Plus (ABI) system using SYBR Premix ExTaq (TaKaRa). PCR reactions were carried out with triplicate. The relative expression of target genes was normalized to the internal reference gene ACTIN (Unigene33024) [59 (link)] using the 2^−ΔΔCT method. Primers are listed in Table S1.

Young leaves pooled from 12 T2 plants of TC-15 and TC-31 were used to analyzed the expression levels of the Cas9 by performed the real-time quantitative PCR (RT-qPCR). PCR primers specific to these genes were designed using PrimerQuest Tool (https://sg.idtdna.com/Primerquest/Home/Index) (Table S8). The total RNA was extracted using the RNAprep Pure Plant Kit (Tiangen, Beijing, China) and reverse-transcribed using the protocol of cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). All RT-qPCR experiments were performed with Applied Biosystems 7900 instruments. The PCR consisted of 10 μL of FastSYBR Mixture (High ROX), 1 μL of forward and reverse primers (10 pM/μL), 7 μL of distilled water, and 1 μL of cDNA in a total volume of 20 μL. The thermal cycling conditions were 3 min at 95 °C followed by 40 reaction cycles (10 s at 95 °C and 20 s at 60 °C). The gene ACTIN2 was used as the reference gene. The mean quantification cycle value of the triple reactions was used to calculate the relative expression levels of the target genes according to the 2^−ΔCt method.

The validation of the RNA-seq analysis was performed by RT-qPCR. After RNA isolation and DNase I treatment, reverse transcription was done with cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). RT-qPCR was carried out with a TaqMan PCR master mix (Thermo Fisher, USA). Normalization was performed against the 16S rRNA gene using the 2^−ΔΔCt method.

Quantitative real-time RT-PCR (qRT-PCR) analysis for validating the different expression data was performed independently under the same conditions described above. First-strand cDNA was synthesized with an oligo(dT) primer using cDNA Synthesis SuperMix (TransGen Biotech, China). Quantitative real-time RT-PCR was carried out in a CFX96 real-time PCR detection system (Bio-Rad) with iTaq Universal SYBR Green Super Mix (Bio-Rad). PCR amplification was performed under the following conditions: 95°C for 3 min, followed by 55 cycles of 95°C for 15 s, 56°C for 15 s, and 72°C for 20 s. Melt curve profiles were analyzed for each gene tested at the end of each PCR. The ubiquitin genes of S. sclerotiorum (SS1G_11035) and B. napus (UBC21) served as the internal reference genes (79 (link), 80 (link)). Primers for the target genes were designed using Beacon Designer V7.92 and are listed in Data Set S1, tab 11.

ZO-1, Occludin and Claudin-1 mRNA expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Total RNA was extracted from the mouse colon using Trizol Reagent (Invitrogen Life Technologies, Grand Island, NY, catalog no. 15596026), and total RNA was reverse transcribed using TransScript First-Strand complementary DNA (cDNA) Synthesis SuperMix (TransGen Biotech, Beijing, China, catalog no. AT301), according to the manufacturer’s instructions. The PCR primers were designed according to the NCBI database (Table). PCR was performed in a StepOnePlus Real-Time PCR System (Oxoid, Thermo Fisher Scientific, MA, USA) using a SYBR Premix Ex Taq (Takara Bio, Japan, catalog no. RR42LR). Each sample was examined in triplicate, and normalized to β-actin with the 2^−ΔΔCT method [20 (link)].

Three biological replicates and three technical replicates were used to validate differentially expressed and differentially spliced genes. Oligo-dT primers and reverse-transcriptase M-MLV were used to treat the total RNA with DNase I, according to the instructions of the manufacturer. A total of 500 ng of RNA was used from each sample, for which reverse transcription was performed with a TransScript^® one-step gDNA removal kit and a cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). A 10 μL reaction volume was used to perform RT-PCR, and a primer pair was designed for each gene to increase each splice variant (isoforms 1 and 2) within a reaction. An Analytik Jena real-time PCR detection system (Analytik Jena AG, Jena, Germany) was used to perform qRT-PCR; the control was the maize actin gene. Table S1 displays the primers used in the qRT-PCR and RT-PCR analyses.