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. 2020 Aug 24;19:128. doi: 10.1186/s12943-020-01246-x

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© The Author(s) 2020

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 5 — circSMARCA5 interacts with its site of transcription. a circSMARCA5 interacts with the 15-16 exon sequence of the SMARCA5 locus. A series of exon DNA fragments were hybridized with circSMARCA in vitro. The DNA-RNA hybridization strength was quantified by dot-blot with R-loop-specific S9.6 antibody. Hybridization stringency was altered by decreasing ionic strength (80-0.1 mM NaCl). b DRIP-qPCR analysis of the 15-16 exon sequence of SMARCA5 to detect the association of circSMARCA5 in MCF-7 cells. RNase H-treated and/or DRIP-qPCR analysis of the 15-16 exon sequence as a control. c CircSMARCA5 partially localized at its site of transcription. Double FISH of circSMARCA5 (red) and its parent DNA region (green). The nucleus was stained by DAPI. d Dot-blot of R-loops in MCF-7 cell genomic DNA preparations treated with DNase I, RNase H, or RNase R. The DNA-RNA hybrids in genome DNA were analyzed by S9.6 antibody. e Mapping of the R-loop formation region of circSMARCA5. A series of exon 15-16 deletion mutants were hybridized with circSMARCA5 for the dot-blotting assay. The DNA-RNA hybridization intensity was analyzed by dot-blot with an S9.6 antibody targeting the DNA-RNA hybrid strand. Hybridization stringency was altered by decreasing ionic strength (80-0.1 mM NaCl)