DOI: 10.25387/g3.28836872.v1
title: Supplemental Material for Grossman <i>et al</i>., 2025

description: RNA sequencing
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Cultures were grown by inoculating conidia from strain JHH-5317 into 25 ml of RPMI to a concentration of 107 conidia/ml, incubating the flasks at 37°C in 5% CO2 to facilitate germination for four hours, then shaking them for 13 h at 37°C in atmospheric conditions. Following this, 25 ml of RPMI containing 16 mg/ml of ITR, 16 mg/ml of VRC, 8 mg/ml of AMB or 2% DMSO by volume were added to each flask, for final concentrations of 8 mg/ml ITR, 8 mg/ml VRC, 4 mg/ml AMB or 1% DMSO. Flasks were shaken at 37°C for two hours, then strained through Whatman #2 filters (Cytiva, Marlborough, MA), washed twice with sterile Milli-Q water, added to 2 ml of Trizol Reagent (Thermo Fisher Scientific, Waltham, MA), flash-frozen and stored at -80°C. Three biological replicates were performed. All drugs were obtained from MilliporeSigma (Burlington, MA) and dissolved in DMSO.
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Cell homogenization was performed in Trizol Reagent using silica spheres (Lysing Matrix C, MP Biomedicals, Irvine, CA) in a FastPrep 120 (MP Biomedicals), with 4 intervals at speed 6 for 30 sec. Homogenates remained on ice between shakes. Total RNA was purified using a PureLink RNA Mini Kit (Thermo Fisher Scientific), with the on-column PureLink DNase treatment, according to manufacturer’s instructions. RNA was quantified using a NanoDrop 1000. Quality assessment was performed by RNA ScreenTape Analysis in a TapeStation 2200 (Agilent Technologies, Santa Clara, CA).
Libraries for RNA-seq were prepared from 250 ng Total RNA using the Illumina TruSeq Stranded mRNA Library Prep kit, according to manufacturer’s Low Sample protocol (Illumina, San Diego, CA). Quality assessment of libraries was conducted by High Sensitivity ScreenTape analysis on a TapeStation 2200 (Agilent Technologies). Libraries were quantified by qPCR with the Kapa Library Quantification kit (Roche, Basel, Switzerland) in a StepOne Plus Real Time PCR System (Thermo Fisher Scientific). Libraries were diluted and pooled and a final quality assessment was performed using High Sensitivity DNA LabChip Analysis on a BioAnalyzer 2100 (Agilent Technologies). A paired end, 2 x 100bp, Illumina HiSeq 2500 run was performed at Johns Hopkins Genomics Genetic Resources Core Facility, RRID:SCR_018669. 
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Genome annotation
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RNA sequencing reads were aligned to each of the three genome assemblies using HISAT2 version 2.2.1 (Kim et al. 2019) and these alignments were input to Stringtie (Pertea et al. 2015) to produce transcript assemblies. We then aligned a collection of 8390 Microascaceae family proteins available from Genbank, to each genome with NCBI tblastn version 2.13.0+, clustered the alignments and produced preliminary CDS features based on the local protein alignments with exonerate in protein2genome mode. We then filtered and reconciled the transcript and protein alignment features with gffcompare and gffread tools to produce final annotation in the GFF format. We then output the protein sequences and aligned them to the proteins in the UniProtKB database with blastp looking for a single best hit (accessed 01/2024). Any protein that had a significant (e-value &lt;10^-8) hit was annotated as being similar to that protein in UniProtKB, and its function was assigned in the GFF file. BUSCO v5 was run on transcripts using gVolante (accessed 02/2024) (Humann et al. 2019; Manni et al. 2021).