Fig. 2: Identification of lncRNA candidates for functional characterization by antisense oligonucleotides (ASOs).

a Diagram showing the selection criteria for the final LEC and BEC lncRNA candidates: (1) sequence conservation of transcription initiation regions (TIR) and/or exon regions; (2) overlap between transcription start sites (TSSs) and DNase hypersensitive sites (DHSs) as a hint for active transcription; (3) expression level cutoffs between LECs and BECs. LEC lncRNAs: TPM and CPM in BECs < 5; TPM and/or CPM > 10 in LECs; BEC lncRNAs: TPM and CPM in LECs < 5; TPM and CPM > 10 in BECs. b, c Heat maps based on expression levels of RNA-Seq (b, TPM, two replicates) and CAGE-Seq (c, CPM, two replicates) of 5 LEC (green) and 12 BEC (red) lncRNAs filtered from a. Color code for row Z score values on a scale from −1 to +1. Genes were ordered by RNA-Seq or CAGE-Seq log2FC values. d, e Expression levels of the two LEC d and two BEC e final lncRNAs in LECs and BECs derived from neonatal and adult donors. Bars represent FC against average BEC or LEC expression. f Representative flow cytometry plots showing the gating strategy used to isolate LECs and BECs from three donors of healthy human skin samples. LECs: CD31+ and CD34−; BECs: CD31+ and CD34+. g Expression levels of the four lncRNA candidates and endothelial (CD31), lymphatic (PROX1, PDPN), and blood (FLT1) markers in freshly sorted LECs and BECs derived from three donors of healthy human skin samples. Bars represent FC values against BEC. h, i Subcellular localization of the two LEC h and two BEC i lncRNAs in neonatal LECs and BECs derived from three donors. Bars represent percentages of nuclear (black) and cytoplasmic (green: LEC; red: BEC) fractions. Data are presented as mean values + SD (n = 3 in d, e, g, h, and i; n = 5 in d and e). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant, n.d.: not detected using paired two-tailed Student’s t test on ∆Ct values against BEC (d, e, and g) or LEC (d, e).