*2.7. Quantitative Real-Time PCR Validation*

Representative differential expressed genes (DEGs) identified by RNA-Seq were selected for experimental quantitative real-time PCR (qRT-PCR) validation. Gene-specific

primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Beijing, China) (Table S1). The RNA was reverse transcribed into complementary DNA (cDNA) using a PrimeScript™ RT reagent Kit with gDNA Eraser (Takara Bio Inc., Kusatsu (Shiga), Japan). qRT-PCR was performed using a TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio Inc., Kusatsu (Shiga), Japan) under the following conditions: 95 ◦C for 30 s, followed by 40 cycles of 95 ◦C for 5 s and 60 ◦C for 30 s. Relative gene expression was calculated using the ΔΔCt method and normalized to the expression levels of *α-tubulin*. The qRT-PCR experiments were performed using at least three biological replicates, a negative control, and two technical replicates.
