*2.2. RNA-Seq Analysis and Quantitative Real-Time PCR Analysis*

Transcriptome sequencing was performed on samples from four treatments—CK, Spd, LF and LF + Spd—collected on day 10 of treatments by Hangzhou Lianchuan Biological Technology Co., Ltd. RNA-seq was performed with three biological replicates for each treatment. All raw sequencing data from the current study were deposited into the NCBI database under the accession number "PRJNA834903" (https://www.ncbi.nlm.nih.gov/ sra/PRJNA834903), (accessed on 4 May 2022). Analysis of significant differences between samples was performed using R packages edgeR or DESeq2. Genes with differential fold FC > twofold or FC < 0.5-fold and a *p*-value < 0.05 were defined as differentially expressed genes [23]. GO (Gene Ontology) enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment were analyzed using the clusterProfiler R package. GO functional enrichment and KEGG pathway analysis were performed by Goatools (https://github.com/tanghaibao/Goatools), (accessed on 6 June 2022) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do), (accessed on 6 June 2022). The qRT-PCR test reaction system and primers used for qRT-PCR are shown in Supplementary Tables S1 and S2, respectively. Samples were added to a 96-well plate and then reacted in an Applied Biosystems Quant Studio three real-time fluorescence quantitative PCR system (QuantStudio 3, ThermoFisher Scientific™, Waltham, MA, USA). The qRT-PCR amplification procedure consisted of Stage 1: pre-denaturation, one cycle 95 ◦C, 30 s; Stage 2: PCR reaction, 40 cycles of 95 ◦C for 10 s, 60 ◦C for 30 s, 72 ◦C for 40 s. Relative gene expression was estimated using the 2-ΔΔCt method [24]. qRT-PCR experiments were performed in biological triplicates.
