Firre lncRNA is functionally integrated into an RNA processing gene expression module. (A) Single-molecule RNA FISH for Firre (green) and Ppib housekeeping mRNA (red) 24 h after transfection of AB2.2 ESCs with control (ctr) ASO or a Firre-specific ASO. (B) Firre expression level in ESCs transfected as in A with control or two independent Firre-specific ASOs. Data represent mean and standard deviation of four biological replicates. (C) Normalized poly(A)⁺ read coverage across the Firre locus in ESCs transfected as in A and B. RNA FISH target sites and ASO target sites are indicated. Due to the repeat motif content, ASOs are complementary to more than one site of the Firre transcript. (D) Overview of differentially expressed genes 24 h after Firre knockdown with two ASOs in four biological replicates each (FDR < 0.05; see Supplemental Fig. S9 for the full heatmap). (E) GO enrichment for the genes affected by both ASO treatments. Top five biological process terms are shown based on Fisher's exact test and after redundancy trimming. (F) GO terms as in Figure 3C obtained from the weighted gene coexpression analysis-derived gene module containing Firre. (G) Firre module genes (red: lncRNA; blue: mRNA) plotted as in Figure 4A. Labeled genes correspond to 18 of the 31 genes (indicated in D, excluding Firre) significantly down-regulated in both Firre ASO treatments.