Investigating the transcriptional changes resulting from loss of Ikaros, Aiolos, or lenalidomide treatment. RNA sequencing (RNAseq) was performed in OPM2, MM1S, and H929 cells 72 hours after IKZF1 (IK20) gRNA induction vs EV control. In OPM2 cells RNAseq was also performed after IKZF3 (Ai35) gRNA induction with dox or treatment with 10 μM lenalidomide. A cutoff false-discovery rate of 0.15 and additional criteria of a fold change ≥1.5 and an RPKM (reads per kilobase of exon length per million mapped fragments) ≥1 were employed for calling of differentially expressed genes. (A) Venn diagram depicting overlap of differentially expressed genes after inactivation of Ikaros, Aiolos, or treatment with lenalidomide in OPM2 cells. Upregulated (Ikaros/Aiolos repressed) genes are in red, downregulated (Ikaros/Aiolos activated) genes are in blue. (B) Top 50 shared upregulated genes in OPM2 cells after loss of Ikaros, Aiolos, or lenalidomide treatment. ISGs are highlighted in red. (C) Venn diagram and (D) graph depicting overlap of common upregulated genes on deletion of Ikaros in OPM2, MM1S, and H929 cells. ISGs are highlighted in red. (E-F) Gene set enrichment analysis and barcode plot of differential gene expression in IK20 in (E) H929 and (F) MM1S cells after dox treatment (as in panel A), tested against a curated list of ISGs from published sources (list shown in supplemental Table 6).^14,24,25  The differential gene expression data set is shown as a shaded rectangle with genes horizontally ranked by moderated t statistic. Genes upregulated upon Ikaros loss are shaded pink and downregulated genes shaded blue. The position of individual ISGs is marked on the plot by vertical black lines. P values for the gene set enrichment test are shown above.