Selection and validation of lncRNA targets. (A) Summary of expression, regulation, and conservation features of the top candidate lncRNA modulators of erythropoiesis (see text). Expression: shown are absolute gene expression estimates (FPKM) from RNA-seq of total RNA from erythroid progenitor-enriched fetal liver cells (PROG) and TER119⁺ erythroblasts (ERY) or of poly(A)⁺ RNA from primary megakaryocytes (MEG), quantified as in Figure 3. Regulation: heatmaps represent whether promoter-proximal binding by GATA1, TAL1, of KLF1, analyzed as in Figure 4, is seen in ERY or MEG. Conservation: heatmap represents whether an orthologous region, identified by local alignment and synteny, is found in the human genome (see supplemental Methods for details). (B) Relative abundance of the top lncRNA candidates across 30 mouse primary tissue and cell types from ENCODE, determined as in Figure 2. Color intensity represents the fractional expression level across all tissues examined. ERY_1 and ERY_2 (red) are TER119⁺ fetal liver erythroblast biological replicate experiments. (C) Relative expression of the top lncRNA candidates across mouse organs and cells of different tissues and developmental stages, as determined by qPCR. Expression levels were normalized to those of 18S rRNA, and fold changes were calculated relative to fetal TER119⁺ erythroblast levels. Data are shown as mean ± standard error of the mean (SEM; n = 3). (D) Detection of individual lncRNA transcripts by single-molecule RNA FISH. Shown are maximum z-stack projections of fluoresce microscopy images of fixed TER119⁺ erythroblasts hybridized to singly-labeled RNA FISH probes. lncRNA molecules are pseudocolored red and DAPI-stained nuclei are pseudocolored blue. For each panel, the mean ± SEM (n = 2) percent of nuclear-localized transcripts is shown at the bottom right corner.