Jarid1b knockdown modulates molecular mechanisms implicated in maintenance of stemness. (A) Quantification of Hox gene transcripts in shJarid1b cells and shLuc controls as assessed by RNA sequencing (RNAseq) analysis. For each Hox cluster (A-B) genes, FPKM (fragments per kilobase per million reads) expression values are shown for both conditions. Error bars indicate standard deviation. RNA was isolated from HSC-enriched cells in culture (4 days following retroviral infection), and only cultures with GT rates >90% were selected. For each condition, 2 biological replicates were sequenced. (B) Average FPKM and fold-change expression values of the 40 most upregulated (FPKM >1 for shJarid1b cells) and (C) downregulated (FPKM >1 for shLuc controls) genes from the RNAseq experiment described in Figure. 6A. Genes annotated to specific functions are specified by a cross mark in respective columns. Complete data for all differentially expressed genes (q value <0.05; all FPKM values included) shown in supplemental Table 7. (D) Enrichment for H3K4me3 marks (black peaks) at the Hoxa7 and Hes1 loci in shJarid1b cells. Chromatin immunoprecipitation was carried out using day 7 (Figure 5C) shJarid1b or shLuc-cells. Total H3K4me3 levels are presented in supplemental Figure 6. (E) Top panel: Experimental strategy for generation of Nup98Hoxa10-homeodomain (NA10hd) plus shRNA-overexpressing cells. Following puromycin selection, the Sca1⁺CD43^- Gr1^-F4/80^- NA10hd-transduced cells were infected with shLuc-, shMoz-, and shJarid1b 1b-carrying retroviruses. Lower panel: Jarid1b knockdown suppresses differentiation of NA10hd overexpressing cells. Proportions of Gr1⁺F4/80⁺ (ie, differentiated) cells in cultures were determined by flow cytometry on day 7 after shRNA transduction. Each dot represents individual culture comprising the transduced progeny of 1500 CD150⁺CD48^-Lin^- bone marrow cells¹ : manual curation^2,3 : 3.4- and 3.7-fold enrichment with false discovery rate of 4.6E-24 and 4.2E-11 in Gorilla bioinformatic tool. *Denotes high H3K4me3 densities, refer to Figure 6D.