2500 machine

Cells from frozen biomass samples were disrupted using a TissueLyser LT disrupter (Qiagen), and total RNA was extracted using an RNeasy minikit (catalog no. 74104; Qiagen). DNA libraries for sequencing were prepared from the total RNA using Illumina’s TruSeq protocol and sequenced using an Illumina 2500 machine, yielding 99 nucleotide paired-end reads with an average insert size of 600 nucleotides. Raw RNA-seq reads were mapped to the individual Penicillium genomes using a documented work flow (37 (link)) based on the TopHat2 (version 2.0.9) (38 (link)) and HTSeq (39 (link)) programs. Both programs were run with default parameters. Gene-level statistics for CM versus DM were calculated using the DESeq2 program with default parameters, and differentially expressed genes were identified based on an adjusted P value cutoff of 0.05. For all downstream analyses, log-transformed expression levels were used.

Confluent cells were treated with 1 μM JQ1- or JQ1 for 48 h, before RNA extraction using the RNeasy kit (Qiagen). RNA sequencing was performed on 3 independent experimental replicates for each cell line and treatment. Following polyA-enrichment and library preparation, 50 bp paired-end sequencing (Illumina 2500 machine) generated >25 million read-pairs per sample. Reads were aligned to the human reference genome (GRCh37) using TopHat2^{52 (link)} and duplicate reads removed (Picard Tools MarkDuplicates). ~20 million high-quality reads per sample were mapped uniquely to Ensembl-annotated genes; gene counts were summarised using HTSeq⁵³ and filtered to exclude genes with fewer than 10 reads on average per sample. Data normalisation and differential expression analysis, comparing JQ1 and JQ1- in each cell line separately, was performed using the edgeR package.⁵⁴ Genes with adjusted P-value <0.05 and showing a fold change >2 in either direction were considered significant. Identification of altered cellular pathways was undertaken using QIAGEN Ingenuity Pathway Analysis (IPA, QIAGEN, www.qiagen.com/ingenuity).