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Chromatin modification mapping in nanochannels
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/content/aip/journal/bmf/7/6/10.1063/1.4833257
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http://aip.metastore.ingenta.com/content/aip/journal/bmf/7/6/10.1063/1.4833257
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Image of FIG. 1.

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FIG. 1.

Schematic of experiment. Chromatin is reconstituted with histone mixtures from different sources, and will in general display a heterogeneous modification profile. It is double-labeled using antibodies for two distinct histone tail modifications that are not mutually exclusive, and introduced into a device that elongates the chromatin molecule. The pattern of binding yields a modification profile.

Image of FIG. 2.

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FIG. 2.

Fluorescence images of chromatin reconstituted from calf-thymus histones. (a) Visualized using only YOYO-1. Chromatin in (b) is stained with Alexa488 anti H3K4-me3 (green) and YOYO-3 (red), in (c) it was stained using Alexa 568 anti H3K4-me3 (red) and YOYO-1 (green), and in (d) it was stained using Alex 568 anti H3K9-ac (red) and YOYO-1 (green). In panels (b)–(d) we present the combined image (center), and show the color channels above (green) and below (red) for clarity. In all panels the scale bar is 5 m.

Image of FIG. 3.

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FIG. 3.

Fluorescence images of double-stained chromatin with Alexa488-anti H3K4me3 (green channel and top panel) and Alex568-anti H3K9ac (red channel and bottom panel). Chromatin was reconstituted with histone extracted from (a) calf thymus, (b) HeLa cells, and (c) chicken erythrocytes. The scale bar is 5 m.

Image of FIG. 4.

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FIG. 4.

Histograms of pixel brightness within channel. Chromatin was reconstituted from (a) calf thymus histones, (b)HeLa cells, and (c) chicken erythrocytes. The density scale is logarithmic, and the white lines indicate the slope from which the ratio of H3K4me3 to H3K9ac was derived.

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/content/aip/journal/bmf/7/6/10.1063/1.4833257
2013-11-21
2014-04-18

Abstract

We report the simultaneous mapping of multiple histone tail modifications on chromatin that has been confined to nanofluidic channels. In these channels, chromatin is elongated, and histone modification can be detected using fluorescently tagged monoclonal antibodies. Using reconstituted chromatin with three distinct histone sources and two histone tail modification probes (H3K4me3 and H3K9ac), we were able to distinguish chromatin from the different sources. Determined ratios of the two modifications were consistent with the bulk composition of histone mixtures. We determined that the major difficulty in transitioning the mapping method to site-specific profiling within single genomic molecules is the interference of naturally aggregating, off-the shelf antibodies with the internal structure of chromatin.

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Scitation: Chromatin modification mapping in nanochannels
http://aip.metastore.ingenta.com/content/aip/journal/bmf/7/6/10.1063/1.4833257
10.1063/1.4833257
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