Validation of RNA-seq Data by qRT-PCR

To verify the accuracy and repeatability of the RNA-seq data, 15 DEGs were selected for qRT-PCR validation. The total RNA was extracted from the first true leaf on the 14th d after Pi deficiency treatment by using RNAprep Pure Plant Plus Kit (Tiangen Biotech, Beijing, China), and cDNA was synthesized using PrimeScript^TM RT Master Mix. Quantitative RT-PCR was performed using an SYBR^® Premix Ex TaqTM Kit (Tiangen Biotech, Beijing, China) with a Roche LightCycler 96 real-time PCR machine (Roche, Basel, Switzerland) with six replicates. The relative expression was calculated using the 2^−∆∆Ct method [58 (link)]. Actin was used as an internal control [59 (link)]. The primers used for qRT-PCR were listed in Supplementary Table S1.