Fig. 5

RMST is a circRNA in differentiated H9 cells. a Modified UCSC browser representation of mapped read count (black) and back-splice junction count (blue) at the RMST locus in untreated replicate 1 at day 0 and day 45. Back-splice reads span a single junction and only the donor and acceptor exons are indicated. Scales are different in the two time-points to allow visualisation of both. GENCODE (green) and Refseq (purple and grey) annotations are shown, together with genomic scale. Exons within circRMST:E12-E6 are enclosed by a red box, upstream exons highly expressed at day 0 are enclosed by a blue box. b Schematic of RMST exon structure, showing circRNA junction counts (above exons) and canonical counts (below exons) summed across untreated replicates for all time-points (0/45/90). Counts were calculated for all exon combinations within each annotated transcript. Numbers are only shown for junctions with 10 or more supporting reads at 1 or more time-points. Exons specific to a single annotated transcript are colour coded (as in a). Exons present in multiple annotated transcripts are shown in grey. Exons are numbered relative to the longest ENCODE annotation ENST00000538559.6 (ENST~ 559.6). Annotated (red) and inferred (orange) transcription start and stop sites are also shown. Data from IGF-1 treated samples are comparable. c Adapted UCSC screen-shots showing changes in expression at exons of uc001tey.1 and ENST00000538559.6 upstream of E6 (left panel) and miRNA precursors flanking the E12-E6 circRNA (middle and right panels) in all untreated replicates. ENCODE/Broad chromatin state segmentation [52] within the H1 ES cell line is also shown (H1): Light red/bright red/purple = weak/active/poised promoter, light/dark green = weak/elongation transcription, orange/yellow = strong/weak enhancer). Scale is capped at 150 to allow direct comparison between time-points. d-e qPCR confirmation of increase in RMST circRNA and E5-E5B junctions. d. Mean ΔCt values from 3 biological replicates assayed at day 0, 30 and 45 are shown (+/− S.E.M). e ΔΔCt values relative to E12-E13. p-values *p < 0.05, **p < 0.01, ***p < 0.001