## 2 Materials and Methods
### RNA-Seq and Bioinformatics  
RNA-seq libraries were prepared from 500 ng of total RNA using the TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA), following the manufacturer's protocol without any modifications. Library QC and quantification were performed using the High Sensitivity D1000 ScreenTape Assay on the 4200 TapeStation system (Agilent, Santa Clara, CA, USA). Sequencing was performed in a NextSeq 500 sequencing system (Illumina) set to obtain 2 \(\times\) 75 bp reads.  
The sequencing files in fastq format were processed with BBDuk version 39.01 ([[https://doi.org/10.1371/journal.pone.0185056](https://doi.org/10.1371/journal.pone.0185056)]([https://doi.org/10.1371/journal.pone.0185056](https://doi.org/10.1371/journal.pone.0185056)) accessed on 18 August 2023) to filter out adapters and low-quality reads. Quality reports were generated using FASTQC version 0.12.1 ([[https://www.bioinformatics.babraham.ac.uk/projects/fastqc](https://www.bioinformatics.babraham.ac.uk/projects/fastqc)]([https://www.bioinformatics.babraham.ac.uk/projects/fastqc](https://www.bioinformatics.babraham.ac.uk/projects/fastqc)) accessed on 18 August 2023). Read mapping was conducted using STAR software version 2.7.10b against the C. _neoformans_ var. grubii H99 genome. To generate the count table, read counts were quantified with the feature Counts program version 2.0.6 ([[https://doi.org/10.1093/bioinformatics/btt656](https://doi.org/10.1093/bioinformatics/btt656)]([https://doi.org/10.1093/bioinformatics/btt656](https://doi.org/10.1093/bioinformatics/btt656)) accessed on 18 August 2023). Differentially expressed genes (DEGs) were identified using DESeq2 ([[https://doi.org/10.1186/s13059-014-0550-8](https://doi.org/10.1186/s13059-014-0550-8)]([https://doi.org/10.1186/s13059-014-0550-8](https://doi.org/10.1186/s13059-014-0550-8)) accessed on 18 August 2023), applying an adjusted \(p\)-value cutoff of 0.05 and a log2 fold change threshold of 1. Annotation data were retrieved from the FungiDB database ([[https://doi.org/10.3390/jof4010039](https://doi.org/10.3390/jof4010039)]([https://doi.org/10.3390/jof4010039](https://doi.org/10.3390/jof4010039)) accessed on 18 August 2023) and EnsemblFungi ([[https://fungi.ensembl.org/Cryptococcus_neoformans_var_grubii_h99_gca_000149245](https://fungi.ensembl.org/Cryptococcus_neoformans_var_grubii_h99_gca_000149245)]([https://fungi.ensembl.org/Cryptococcus_neoformans_var_grubii_h99_gca_000149245](https://fungi.ensembl.org/Cryptococcus_neoformans_var_grubii_h99_gca_000149245)) accessed on 18 August 2023). The R 4.1 and Bioconductor 3.16 package GOstats ([[https://doi.org/10.1093/bioinformatics/btl567](https://doi.org/10.1093/bioinformatics/btl567)]([https://doi.org/10.1093/bioinformatics/btl567](https://doi.org/10.1093/bioinformatics/btl567)) accessed on 18 August 2023) was used to perform the Gene Ontology (GO) enrichment analysis, and pathview ([[https://doi.org/10.1093/bioinformatics/btt285](https://doi.org/10.1093/bioinformatics/btt285)]([https://doi.org/10.1093/bioinformatics/btt285](https://doi.org/10.1093/bioinformatics/btt285)) accessed on 18 August 2023) was used for the KEGG analysis.