Fig 1: EWS-WT1 loss leads to large-scale gene expression changes in DSRCT cells.a Hierarchical clustering of gene expression changes following silencing of EWS-WT1 in JN-DSRCT-1 cells (JNDSRCT1) or BER cells (BER) relative to a non-targeting control siRNA in the same cells (JN_control or BER_control). Data represent 1879 gene expression changes that are either increased (red) or decreased (blue) by a log FC of >2. b Volcano plots showing the magnitude of gene expression change as a function of q value (false discovery rate-adjusted P value) with silencing. Gene expression changes are both common (red) across the two cell lines and unique to the cell line (blue). More genes are repressed by EWS-WT1 than are induced. Libraries were prepared from 500 ng of total RNA using the KAPA-stranded mRNAseq Kit (v5.17). RNA was sheared to 300–400 bp. Prior to PCR amplification, cDNA fragments were ligated to Bio Scientific NEXTflex dual adapters. Quality and quantity were determined using a combination of Agilent DNA High Sensitivity chip, QuantiFluor® dsDNA System, and Kapa Illumina Library Quantification qPCR assays. Individually indexed libraries were pooled, and 75-bp, paired-end sequencing was performed on an Illumina NextSeq 500 sequencer using a 75-bp HO sequencing kit (v2). Base calling was done by Illumina NextSeq Control Software (NCS) v2.0 and demultiplexed to FastQ format with bcl2fastq v1.9.0 (Illumina Inc.). Reads were aligned to hg38 using STAR (v2.5.2b) with options –twopassMode Basic –quantMode GeneCounts. The data were filtered for a minimum of ten counts per million in at least one sample. Differential expression analysis was carried out using Deseq2 (v1.24.0) with apeglm (v1.6.0) applied to account for absolute magnitude of gene expression. Significant genes were determined using a cutoff of q value < 0.0511. Heatmaps were generated using pheatmap package (v1.0.12) in R (v3.6.1). Analysis was performed on three biological replicates of each sample. All samples were collected at the same time, and RNA was isolated on the QiaCube (Qiagen) in batches after sample randomization to avoid batch effect. The defined EWS-WT1 gene signature consists of genes with a log-fold change of 2 or greater in both cell lines following silencing. c Western blot of EWS-WT1 silencing confirms repression (ERG) and induction (CEBPD) of targets. Lysates collected at 16, 24, 30, and 48 h of exposure. Western blot probed with EWSR1 (11910, Cell Signaling) and H3 (2650, Cell Signaling), WT1 (sc-7385 Santa Cruz Biotechnology), ERG (ab92513, Abcam), and CEBPD (ab65081, Abcam) antibodies.