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. 2018 Mar 1;8:3850. doi: 10.1038/s41598-018-21806-y

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© The Author(s) 2018

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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DSB-dependent deletions are unaffected by modulation of transcription: (A) I-SceI-dependent increase in deletions seven days after I-SceI induction in cells treated with the transcription inhibitor DRB compared to control cells (n = 7). (B) Schematic representation of the transcription regulation system. Upper panel: two TetO cassettes are inserted in front of TetR-IRES-NeoR. Lower panel: experimental design to study deletions in a context of high (+Dox) or low (−Dox) transcriptional activity, indicating I-SceI transfection and induction, doxycycline treatment and FACS analysis. (C) Immunoblot analysis of the cell line treated with or without doxycycline for six days, using antibodies against TetR, GFP and tubulin (as a loading control) (left panel). Right panel: relative quantification (n = 2). (D) TetR mRNA quantification by RT-qPCR, normalized to GAPDH mRNA level (n = 4). (E) Percentage of GFP-positive cells after I-SceI induction in high (+Dox) or low (−Dox) transcriptional activity context (n = 3). (F) DRIP-qPCR analysis of DNA:RNA hybrid structure at TetR-IRES-NeoR gene in undamaged cells (no I-SceI induction). Primers targeting the APOE gene are used as a positive control for R loop formation, and primers specific to an intergenic region are used as a negative control. The values, corresponding to the signal following S9.6 IP of isolated DNA (dark grey bar) or of in vitro RNaseH-treated DNA (clear grey bar), are represented as fold increase normalised to the APOE positive control (n = 7).