(A) Venn-diagram of peak call overlaps for HL-1 ATAC-sequencing (ATAC.HL1), histone 3 lysine 27 acetylation in mouse heart (H3K27ac.Heart, GSE52123), DNase hypersensitivity in mouse heart (DNase.heart, ENCODE) and TBX5 ChIP-seq (TBX5.HL1, GSE21529). (B) Workflow for identifying TF-dependent ncRNAs. Total noncoding transcripts from mouse left atrium were narrowed to Tbx5-dependent distal intergenic ncRNAs, defined as >1 kbp away from the known transcriptional start sites of known coding genes (GENCODE mm10), and narrowed again to coherent changes with nearby Tbx5-dependent genes within a 2-Mbp window. (C). Heatmap of identified Tbx5-dependent ncRNAs in Tbx5fl/fl;R26CreERt2 (red, left) and corresponding R26CreERt2 control (blue, right) in left atrial tissue. The hierarchical cluster analysis is based on the Euclidean distances of normalized sequencing counts. 1577 Tbx5-activated ncRNAs were downregulated after Tbx5 deletion and 1490 Tbx5-repressed ncRNAs were upregulated after Tbx5 deletion across n = 5 and n = 3 resp. (D) Volcano-plot of significantly misregulated TF-dependent ncRNAs, select identifications were labeled by nearest TBX5-dependent gene. Plot of log2 fold-change of ncRNAs in Tbx5fl/fl;R26CreERt2 compared to R26CreERt2 vs –log10 false discovery rate (FDR) for the same comparison (FDR < 0.05, |FC| > 2). The ncRNAs within 2 Mb of coherently mis-expressed TBX5-dependent genes are red or blue for activated and repressed, respectively. Gray dots represent those ncRNAs without coherently mis-expressed coding genes in the 2-Mb window. (E, F) Example genomic views of two of the most significantly TF-dependent ncRNAs, adjacent to the Atp2a2 (E) and Ryr2 (F) genes, respectively. Top track is chromosomal location, followed by the ncRNA read density from R26CreErt2control and Tbx5fl/fl;R26CreERt2. Below is ATAC-Seq peak call in HL-1 cells, cardiac DNase hypersensitivity (ENCODE), TBX5 ChIP-seq (GSE21529) and cardiac H3k27 acetylation (GSE52123). The identified differential ncRNA is marked in the red box, and the putative regulatory element, as defined by the enhancer marks above, is marked in the gray box.