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. 2017 Oct 18;7:13459. doi: 10.1038/s41598-017-13887-y

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© The Author(s) 2017

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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RNA-sequencing analysis of three H3.3^WT and three H3.3^G34W primary cell lines show distinct splicing aberrations. (a) Electropherogram of MNase assay estimating chromatin compaction of mutated and unmutated primary cells. Plot based on 60 min incubation with MNase of H3.3^WT (blue line) and H3.3^G34W (red line) primary cell lines and BioAnalyzer data collection. (b) RNA splicing events of H3.3^G34W vs. H3.3^WT primary cells, extracted by the rMATS and SpliceR algorithms. The increased splicing events in H3.3^G34W when compared to H3.3^WT are given in [%]. (c) Plot of the significant increase of potentially novel isoforms. The data is based on the Cufflinks category j that calls for potentially novel isoforms (fragments) with at least one splice junction shared with a reference transcript. (d) Three examples (AURKA, NASP, and TPM2) of exon inclusion events in H3.3^G34W, first observed in RNA-seq. and here validated with the nCounter RNA-based hybridization technology. (e) Exon skipping in TUFT1 occur in H3.3^G34W, and probe detects junction of flanking exons to the skipped exon. (f) Alternative transcriptional start sites at the DDX10 and TPM1 locus. Increased read counts in their first or second introns indicated by red boxes. Plot of nCounter validations are shown in the right panel, where loss of normal start correlates with gain of alternative start.