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. 2020 Dec 7;8:219. doi: 10.1186/s40478-020-01092-4

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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. 1 — Genetic Modification of Histone H3.3 expression in Pediatric Glioma Cells and Astrocytes. a Expression of RFP reporter was confirmed in > 95% of Doxycycline-induced cells. b H3.3G34V expression was significantly reduced in lentiviral-transduced KNS42 cells treated with Doxycycline, compared to no Doxycycline and control conditions. **p < 0.01, ****p < 0.0001. c H3.3G34V protein was expressed in lentiviral-transduced NHAs treated with doxycycline, with no H3.3G34V expression observed in controls. d Flow cytometry was used to select the top 30% cells with H3.3G34V knockdown and knock-in for subsequent ChIP-Seq analysis. e Genome browser view of H3.3G34V enrichment at the H3F3A locus across cell lines studied. Tracks highlighted in red are those conditions without H3.3G34V expression. f Metagene profile of H3.3G34V enrichment in the gene body across the top 1000 most H3.3G34V-enriched genes in KNS42. Reduction of H3.3G34V enrichment is observed following doxycycline-induced knockdown (light blue versus navy blue lines). Greater H3.3G34V enrichment is also observed in NHA transduced for H3.3G34V expression (green line) compared to control transduced for H3.3WT expression (orange line)