RNA sequencing

For the identification of transcriptional changes (Fig. 3, C to F) in aRMS cells after ENT treatment, each of the three aRMS cultures (human cell lines Rh30 and Rh41 and mouse cell culture U23674) was treated with ENT alongside a paired untreated sample for a fixed time period. All samples were treated with ENT for 72 hours, except for Rh41, which was treated for 24 hours because of higher sensitivity to ENT. All cells were cultured on 10-cm dishes, and treatment began when plates were 60% confluent. Passages lower than 7 were used for all mouse cultures. RNA isolation and sequencing were performed by the commercial service provider, Beijing Genomics Institute (BGI). Differential expression for a single sample was defined as post-ENT treatment expression divided by vehicle-treated expression. These criteria identified 348 overexpressed and 358 underexpressed genes in aRMS. Differential expression of genes associated with chromatin modification and remodeling after ENT and PAN treatment (72 hours) (Fig. 5A) was analyzed by one more set of RNA-seq with RNA isolation and sequencing performed by BGI. Log₂ scaled ratios of ENT-treated versus vehicle-treated RH30 and U23674 and PAN-treated versus vehicle-treated RH30 and U23674 were organized by change in regulation.