## 3 Results  
### Identification of Enhancer RNAs Expressed in Erythroblasts Involved in Long-Range Interactions  
To identify erythroid-specific enhancer RNAs (eRNAs) with long-range interactions, we analyzed previously published data by using the strategy outlined in Figure 1A. We used RNA-seq data from a mouse E14.5 fetal liver [24; 49; 50], which is essentially an erythroid organ, and assembled the transcriptome using StringTie [69]. To identify novel eRNA transcripts, we applied GffCompare to the assembled transcriptome, removed transcripts overlapping known protein-coding genes, and further selected transcripts with class codes \"x\", \"u\", \"l\", and \"r\". We then selected transcripts overlapping mouse erythroid enhancers using Bedtools [65]. The mouse erythroid enhancer dataset employed is a union of enhancers from ENCODE3 mouse E14.5 fetal liver cells [57] and enhancers annotated in MEL, G1E, and G1 ER erythroid cell lines by EnhancerAtlas 2.0 [58]. Transcripts with coding potential were removed using the Coding Potential Calculator (CPC2) [51], and as a result, 972 eRNAs were identified.  
We then used published Promoter Capture HiC data from mouse E14.5 fetal liver cells [52] to select from these 972 eRNAs those with long-range interactions (>50 kb), obtaining 791 such eRNAs. To further identify erythroid-specific long-range interacting eRNAs, we analyzed the tissue/cell expression patterns of these 791 long-range interacting eRNAs using 28 total RNA-seq datasets and 56 polyA RNA-seq datasets from ENCODE with classification performed using TissueEnrich [55]. The resulting 82 erythroid-specific  
Figure 1: Identification of enhancer RNA expressed in the erythroblast with long-range interactions. (**A**) Workflow showing the discovery of enhancer RNAs in erythroid cells with long-range interactions, published data from [24; 49; 50; 52] were used for analysis. (**B**) Heatmap showing the expression of the long-range interacting erythroid enhancer RNA in BFU, CFU, and Ter119-positive cells from mouse E14.5 fetal liver. RNA-seq data from [PERSON], et al. [50]. (**C**) Gene ontology analysis of the target genes that interact with Ter119 cells’ specific long-range interacting erythroid enhancer RNAs.  
eRNAs commonly identified among all datasets were defined as erythroid-specific long-range interacting eRNAs.  
We next examined the expression patterns of these erythroid-specific long-range interacting eRNAs across erythroid lineages, including burst-forming units (BFU), colony-forming units (CFU), and Ter119+-maturing erythroblasts [50]. The majority of eRNAs were induced in erythroblasts, suggesting a role in terminal erythropoiesis (Figure 1B). We performed differential expression analysis with DEseq2 and identified 16 eRNAs that were significantly upregulated in Ter119+ cells. Gene ontology analysis of the 53 genes interacting with these 16 eRNAs loci shows enrichment for the heme biosynthetic process (Figure 1C). These results show that while a few of the erythroid eRNA loci with long-range interactions that we identified as being upregulated in late erythropoiesis make contact with erythroid genes, many do not. This implies that if these eRNAs function in late erythropoiesis, they may act indirectly or regulate erythroid genes in trans without relying on chromatin looping.