Primescript rt reagent kit with gdna eraser

To verify the accuracy of transcriptome data, quantitative reverse-transcription PCR was performed on 9 DEGs altered after Agrobacterium infection. The specific primers were designed using Primer 6.0 (Supplementary Table S5). First-strand cDNA was synthesized from 0.5 μg of total RNA, as mentioned above, using the PrimeScript^TM RT reagent kit with gDNA Eraser (Takara, Dalian, China), according to the manufacturer’ s instructions.
The synthesis of first-strand cDNA was performed using the PrimeScript^TM RT reagent kit with gDNA Eraser (Takara, Dalian, China) according to the manufacturer’ s instructions. Quantitative reverse-transcription PCR was conducted using the QuantStudio 3 Real-Time PCR system (Thermo Fisher, Waltham, MA, USA) with TB Green™ Premix Ex Taq™ II (TaKaRa, Dalian, China). The programmer performed the following steps: 10 min at 95 °C, followed by 40 cycles of 5 s at 95 °C and 31 s at 55 °C. The actin gene was employed as the reference gene for the experiments (Supplementary Table S5). All of the samples were examined in triplicate, with two technical replicates. The relative expression of these genes was calculated by the 2^−ΔΔCt method [67 (link)]. Subsequently, the gene expression data were graphically screened on the bar chart with Microsoft Excel 2016.

The cDNA was synthesized via reverse transcription using the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa). Then, the qRT-PCR was performed using TB Green^® Premix Ex Taq™ II (Tli RNaseH Plus) (TaKaRa) on an ABI ViiA 7 system. The results were analyzed using the relative quantitative method, and the mRNA expression of genes was normalized with Gapdh. The primers for qRT-PCR are shown in Table S1.
A strand-specific reverse transcriptase reaction was performed using the method a previous study reported [67 (link)] for the detection of paternally expressed Rtl1 using the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa). The PCR results were quantified with Image J 1.53e software (Wayne Rasband and contributors National Institutes of Health, USA, http://imagej.nih.gov/ij), and the results were normalized to Gapdh expression.

The first-strand cDNA was synthesized from 1 μg RNA using a Prime ScriptTM RT reagent kit with gDNA Eraser (TaKaRa, Kusatsu, Shiga, Japan) based on the manufacturer’s protocol. The cDNA was synthesized from 1 μg of total RNA with PrimeScript™ RT reagent Kit with gDNA Eraser following the manufacturer’s instructions (Takara, Japan). For quantitative reverse transcription polymerase chain reaction (qRT-PCR), relative gene expression levels of the target gene were measured using a Roche LightCycler480 RT-PCR system (Roche, Swiss). The SYBR Green real-time PCR mix was added to the reaction system according to the manufacturer’s instructions. The primers were designed using Primer premier 6 based on Cucurbit Genomics Database (http://cucurbitgenomics.org/) and listed in Additional file 1: Table S13. All genes were run in triplicate from the three biological replicates. The raw data of qRT-PCR were analyzed using LCS 480 software 1.5.0.39 (Roche, Swiss) and the relative expression was determined by using the 2^−ΔΔCT method. The watermelon ClACTIN genes were used as internal control [57 (link)].