## 2 Materials and Methods
### Sample Preparation and Analysis of Metabolism
#### 2.4.5 Transcriptome and Metabolomic Assembly and Annotation  
To ensure high-quality and accurate data, the FASTA software was used to filter out unnecessary reads from the original information [22]. The chosen data was then converted into a transcriptome, and the unigene function was annotated using various databases, including non-redundant protein sequences from the NCBI (National Center for Biotechnology, [[https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/](https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/)]([https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/](https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/)), accessed on 12 December 2021), KEGG (Kyoto Encyclopedia of Genes and Genomes, [[https://www.genome.jp/kegg](https://www.genome.jp/kegg)]([https://www.genome.jp/kegg](https://www.genome.jp/kegg)), accessed on 12 December 2021), COG (Clusters of Orthologous Groups of proteins, [[https://www.ncbi.nlm.nih.gov/COG/](https://www.ncbi.nlm.nih.gov/COG/)]([https://www.ncbi.nlm.nih.gov/COG/](https://www.ncbi.nlm.nih.gov/COG/)), accessed on 12 December 2021), GO (Gene Ontology, [[https://www.geneontology.org](https://www.geneontology.org)]([https://www.geneontology.org](https://www.geneontology.org)), accessed on 12 December 2021), Swiss-Prot ([[http://www.ebi.ac.uk/uniprot/](http://www.ebi.ac.uk/uniprot/)]([http://www.ebi.ac.uk/uniprot/](http://www.ebi.ac.uk/uniprot/)), accessed on 12 December 2021), KOG (Karyotic Orthologous Groups, [[https://www.ncbi.nlm.nih.gov/KOG/](https://www.ncbi.nlm.nih.gov/KOG/)]([https://www.ncbi.nlm.nih.gov/KOG/](https://www.ncbi.nlm.nih.gov/KOG/)), accessed on 12 December 2021), and Pfam (Protein family, [[https://www.Pfam.janelia.org](https://www.Pfam.janelia.org)]([https://www.Pfam.janelia.org](https://www.Pfam.janelia.org)), accessed on 12 December 2021).  
4.6 Expression of Triterpeneoid Biosynthesis-Related Genes in \(S\). _sanghuang_ under Elicitor Treatment  
Based on the studies conducted by [PERSON] et al. [23] and [PERSON] et al. [24], three unigenes associated with secondary metabolic biosynthesis were selected and verified using qRT-PCR. The RNA was reverse transcribed into cDNA using a TUREscript 1 st Stand cDNA Synthesis Kit (Aidlab, Qingdao, China). The qRT-PCR was conducted on a German Analytik Jena-qTOWER 2.2 real-time PCR system, using 2\(\times\) SYBR Green Premix (Qingdao, China). Six biological repeats and three technical repeats were performed, and the names and sequences of the primers used in all experiments are provided in Table S2. For each reaction, a 10 \(\mu\)L volume was used, which contained SYBR Premix Ex TaqMan II (3 \(\mu\)L), forward primer (200 nM, 0.5 \(\mu\)L), reverse primer (200 nM, 0.5 \(\mu\)L), cDNA template (5 ng/\(\mu\)L, 1 \(\mu\)L), and ddH2O (3 \(\mu\)L). The qPCR parameters included an initial step of 95 \({}^{\circ}\)C for 3 min, followed by 40 cycles of amplification (95 \({}^{\circ}\)C for 10 s, 60 \({}^{\circ}\)C for 30 s). The melting curve was measured after holding at 60 \({}^{\circ}\)C to 95 \({}^{\circ}\)C for 4 s, with an increase of +1 \({}^{\circ}\)C/cycle, using the 18S gene as an internal control reference gene. Finally, the 2\({}^{-\Delta\Delta\text{Ct}}\) method was used to calculate the gene expression [25].