7500 fast and transstart top green qpcr supermix

RNA extraction was used EASYspin Plant RNA Kit (Aidlab Biotech, Beijing, China). RNA reverse transcription used TranScript® II All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) from TransGen Biotech. 1000 ng RNA to reverse transcribe into cDNA, and diluted the cDNA four times for qRT-PCR. The qRT-PCR was performed by using the Gene Applied Biosystems® 7500 Fast and TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). qRT–PCR was conducted in a 20 μl volume containing 1 μl of diluted cDNAs, 0.4 μl of the each primer, 0.4 µL of passive reference dye, 7.8ul H₂O, and 10 µL of Top Green qPCR SuperMix under the following conditions: 95 ◦C for 300 s, followed by 40 cycles of 95 ◦C for 15 s, 58 ◦C for 20 s and 72 ◦C for 30 s. GhActin gene was used as an internal reference gene. The 2^−△△Ct method was used to calculate relative expression levels [37 (link), 38 ].

To verify the reproducibility of DEGs in the RNA-seq data, we used the same samples for evaluating the gene expression levels by qRT-PCR. Ten genes from the DEGs were selected randomly, and corresponding primers were designed by NCBI (Primer-Blast). The gene name and primer sequence of the DEGs are listed in Table S1. Total RNA of the tested samples in RNA-seq reverse transcribed using the PrimeScript™ RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara, Dalian, China). Then, qRT-PCR was performed by using the Gene Applied Biosystems@7500 Fast and TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). Each sample was analyzed with three technical replicates. The PCR cycle was configured as: 5 min at 95 °C followed by 40 cycles of amplification at 95 °C for 15 s, then 58 °C for 20 s, and 72 °C for 30 s. The relative expression level of the DEGs was calculated by the 2^−ΔΔCt [49 (link)]. The GAPDH gene was used as the internal control [50 (link)].