Platform

Substrate

Sample condition

RNA-seq data analysis

Reference

Maxima SYBR Green qPCR Master Mix and ABI apparatus (Applied Biosystem)

Spruce wood, Microcrystalline cellulose (Avicel)

Three biological replicates; total RNA was extracted from fungal mycelia growing on spruce wood at 28˚C at 7, 14, 21, and 28 days; total RNA was extracted from fungal mycelia growing with Avicel cultures at 14 and 28 days.

Differences in the gene expression levels were estimated according to the Shapiro-Wilk normality test at P ≥ 0.05. Normal distribution was done for genes cel7b and lpmo1 based on two biological replicates of fungi growing with Avicel at 14 days. Repeated measures ANOVA was used to estimate the variation in the expression of one gene in different time points for spruce wood cultures and paired samples t-test for Avicel cultures at P-value < 0.05.

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DNBseq Technology

Aspen, spruce wood, wheat bran, cottonseed hulls

Total RNA was extracted from mycelia from two biological replicate cultures after 9 days and 16 days’ cultivation at 28˚C

Reads were mapped to the genome sequence of D. squalensLYAD-421 SS1 (v1.0 annotation, the Joint Genome Institute (JGI)) using SOAPALIGNER/SOAP2. RPKM was used to quantify RNA-seq results, Differential expression was identified by CyberT Bayesian ANOVA algorithm at a cut-off value of the fold change of >1.5 and P-value (corrected by multiple tests) of <0.05.

[40]

Illumina HiSeq 2000 platform

Norway spruce (Piceaabies), silver birch (Betula pendula)

Three biological replicates; total RNA was extracted from mycelium after 2 weeks and 4 weeks cultivation at 28˚C

Reads were aligned to the reference genome (https://genome.jgi.doe.gov/Dicsqu464_1/Dicsqu464_1.home.html) using HISAT version 0.1.4-beta. Differentially expressed genes were identified using DESeq2 (version 1.10.0) with a cutoff

value of adjusted p-value < 0.05. Raw gene counts were used for DGE analysis as DESeq2 uses its internal normalization.

[41]

Illumina HiSeq 2000 platform

D-glucuronic acid, D-galacturonic acid, L-rhamnose, D-galactose,

D-xylose, D-mannose, L-arabinose, disaccharide cellobiose

The replication of samples was not mentioned in this study. Total RNA was extracted from the mycelium from the edge of the colony after 5 days of growth at 28˚C

Reads were mapped to the genome of D. squalens CBS 464.89 using Bowtie2 and the BWA software. The gene expression level was measured in FPKM using the RSEM tool. Differential expression was identified by the DESeq2 with a cutoff value of ≥2.5-fold change, FPKM value of ≥10, and the adjusted P-value of ≤0.01. CAZyme annotations were done using JGI MycoCosm website (https://genome.jgi.doe.gov/mycocosm/proteins-browser/browse;qLeIA4?p=Dicsqu464_1).

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Illumina HiSeq 2500 platform

coniferyl alcohol, ferulic acid, vanillin, vanillyl alcohol, vanillic acid, veratryl alcohol, protocatechuic acid, p-coumaric acid, p-hydroxybenzoic acid, and cinnamic acid.

Three biological replicates; RNA was extracted from cultivation for 4 days at 28˚C.

Gene expression levels were measured as FPKM. DESeq2 version 1.10.0 was used to compare the transcript level of samples. Differentially expressed genes were identified with fold change > 2, adjusted p < 0.01, and FPKM > 10 in at least one condition between each pair of conditions. Functional annotation of differentially expressed genes was based on combined information from EuKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping, InterPro protein sequence analysis and classification, and Carbohydrate-active enzymes (CAZy) classifications for D. squalens CBS464.89 (Dicsqu464_1) retrieved from JGI MycoCosm database (https://genome.jgi.doe. gov/cgi-bin/kogBrowser?db=Dicsqu464_1)

[43]