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. 2020 Jan 3;11:90. doi: 10.1038/s41467-019-13840-9

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a The correlation between simulated and estimated junction ratio by CIRIquant and three other methods. x- and y-axis represent the expected and estimated junction ratio of circRNAs, respectively. For DCC, the junction ratio was calculated using the results from LinearCount and CircRNACount output. For Sailfish-cir, the TPM of circRNAs and corresponding host genes were used for junction ratio calculation. b Schematic view of experimental design for shRNA induced knockdown of three splicing factors (MBNL1, PTBP1, and Tra2B) and the calculation of DE- and DS- score. Specially designed shRNAs were used to knockdown target genes. Then, RNA-seq were performed for control and knockdown groups, and circRNA expression levels and junction ratio were estimated by CIRIquant for further differential expression analysis. c Heatmap of DE score and DS score in three knockdown datasets. Only circRNAs significantly changed in at least one sample are shown in the heatmap. d Overlap of significantly changed circRNAs. x- and y-axis represent −log10(p value) from DE-analysis and rate-ratio test using junction ratio of circRNAs in two different conditions, respectively. Downregulated p-values are assigned as negative values. DE-specific and DS-specific circRNAs are highlighted in blue and red, respectively. e Gene ontology enrichment analysis of differentially expression genes and all significantly changed DE- and DS-circRNAs. The logarithm of FDR was calculated in Enrichr, then plotted in heatmap for comparison. f Junction ratio changes of LC-switching circRNAs after MBNL1/PTBP1/TRA2B knockdown. g Average depth of binding sites in flanking regions of differentially spliced ES exons and significantly changed circRNAs using CLIP-seq results from iCLIPdb. A sliding window analysis with 8 bp windows was performed for density calculation. Vertical axis represents the average binding site per shifted window.