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. 2024 Feb 10;17:3. doi: 10.1186/s13072-024-00527-9

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© The Author(s) 2024

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 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 — Development of an optimised ChIP-reChIP protocol to robustly measure bivalent chromatin. A Potential limitations in using independent total H3K4me3 (green, circles) and total H3K27me3 (red, triangles) datasets in distinguishing bone-fide bivalent chromatin, where the two marks occur on the same nucleosome, from allelic and cellular heterogeneity. B overview of sequential ChIP-reChIP protocol, see methods for detailed description. C Agarose gel showing chromatin digested with 1.2μl, 2.4μl or 4.8μl MNase for different amounts of time (7.5 min to 60 min). 2 million cells were used per condition. Negative control did not have any MNase added. Star denotes final condition used in protocol. D Single H3K4me3 (green) and IgG control (grey) ChIP-qPCR analysis comparing SDS-based elution (light) from peptide elution (dark). Two active H3K4me3-only (Dppa2, Dppa4), two inactive H3K27me3-only (Gm6116, K27me_R1) and four bivalently marked loci (Csf1, Lmo1, Pou4f1, Sox6) were analyzed. Enrichment values normalised to input are shown