RNA-Seq analysis of JQ1 inhibition

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).