Smc3 haploinsufficiency perturbs mRNA transcriptional output. RNA-seq using synthetic RNA spike-in controls reveals RNA transcript levels are globally decreased in Smc3^Δ/+ c-Kit⁺ cells when compared with Smc3^fl/+ WT. (A and B) The widespread decrease in overall mRNA levels is obscured when spike-in control reads are not used (A) but apparent with cell number normalization (B); fold change values represent the mean of duplicate samples. (C) These effects are apparent across gene expression levels; gray line represents gene expression of Smc3^fl/+ cells rank ordered by expression level; red dotted line represents LOESS curve through Smc3^Δ/+ matched values. (D) Scatterplot of genes identified as unchanged and down-regulated with Smc3 haploinsufficiency with exemplary genes in each group shown. Gene lists generated as defined in Materials and methods; full gene lists annotated in Table S1. (E) GSEA using a human hematopoietic progenitor gene set, with several enriched down-regulated genes highlighted. (F and G) Pairwise GSEA of mouse (F) and human (G) hematopoietic cells using the down-regulated gene set. Cumulative enrichment scores represent the sum of enrichment scores for each cell type when compared across all other cell types.