Name: imaging replicative domains in ultrastructurally preserved chromatin by electron tomography
Uploaded: 2022-05-20T14:00:00
Description: Watch this Scientific Journal Video about Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography at JoVE.com

This work was supported in part by RSF (grant #17-15-01290) and RFBR (grant #19-015-00273). The authors thank Lomonosov Moscow State University development program (PNR 5.13) and Nikon Center of Excellence in correlative imaging at Belozersky Institute of Physico-Chemical Biology for access to imaging instrumentation.

The protocol is optimized for adherent cells and was tested on HeLa, HT1080, and CHO cell lines.
1. Cell labeling and fixation
<ol>
	<li>Plate cells on acid-cleaned coverslips in a 3 cm Petri dish. Grow the cells in the media recommended for the cell line being used to 70% confluency.</li>
	<li>Add EdU (5-ethynyl-2&#39;-deoxyuridine) from 10 mM stock to 10 &#181;M final concentration and place the cells in the incubator for 10 min or longer (depending on the experiment objec...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superresolution+imaging+reveals+spatiotemporal+propagation+of+human+replication+foci+mediated+by+CTCF-organized+chromatin+structures.">Superresolution imaging reveals spatiotemporal propagation of human replication foci mediated by CTCF-organized chromatin structures.</a> Proceedings of the National Academy of Science. 117 (26), 15036-15046 (2020).</li><li>Leonhardt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+DNA+replication+factories+in+living+cells.">Dynamics of DNA replication factories in living cells.</a> Journal of Cell Biology. 149 (2), 271-280 (2000).</li><li>Philimonenko, A. A., Hodn&#253;, Z., Jackson, D. 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B., Wu, W., Bian, Q., Kireev, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+interphase+large-scale+chromatin+structure+from+analysis+of+engineered+chromosome+regions.">Insights into interphase large-scale chromatin structure from analysis of engineered chromosome regions.</a> Cold Spring Harbor Symposium on Quantitative Biology. 75, 453-460 (2010).</li><li>Lieberman-Aiden, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+mapping+of+long-range+interactions+reveals+folding+principles+of+the+human+genome.">Comprehensive mapping of long-range interactions reveals folding principles of the human genome.</a> Science. 236 (5950), 289-293 (2009).</li><li>Pope, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topologically+associating+domains+are+stable+units+of+replication-timing+regulation.">Topologically associating domains are stable units of replication-timing regulation.</a> Nature. 515 (7527), 402-405 (2014).</li><li>Ou, H. D., Phan, S., Deerinck, T. J., Thor, A., Ellisman, M. H., O'Shea, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChromEMT:+Visualizing+3D+chromatin+structure+and+compaction+in+interphase+and+mitotic+cells.">ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells.</a> Science. 357 (6349), (2017).</li><li>Hoz&#225;k, P., Jackson, D. A., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+factories+and+nuclear+bodies:+the+ultrastructural+characterization+of+replication+sites+during+the+cell+cycle.">Replication factories and nuclear bodies: the ultrastructural characterization of replication sites during the cell cycle.</a> Journal of Cell Science. 107 (8), 2191-2202 (1994).</li><li>Deng, X., Zhironkina, O. A., Cherepanynets, V. D., Strelkova, O. S., Kireev, I. I., Belmont, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytology+of+DNA+replication+reveals+dynamic+plasticity+of+large-scale+chromatin+fibers.">Cytology of DNA replication reveals dynamic plasticity of large-scale chromatin fibers.</a> Current Biology. 26 (18), 2527-2534 (2016).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Science. 105 (7), 2415-2420 (2008).</li><li>Gilerovitch, H. G., Bishop, G. A., King, J. S., Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+electron+microscopic+immunocytochemistry+with+silver-enhanced+1.4-nm+gold+particles+to+localize+GAD+in+the+cerebellar+nuclei.">The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellar nuclei.</a> Journal of Histochemistry and Cytochemistry. 43 (3), 337-343 (1995).</li><li>Kireev, I., Lakonishok, M., Liu, W., Joshi, V. 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The method described here has several advantages over previously published protocols. First, the use of Click-chemistry for labeling replicated DNA eliminates the necessity of DNA denaturation prerequisite for BrdU detection with antibodies, thus better preserving chromatin ultrastructure. 
Second, utilization of biotin as a secondary ligand that is generated after glutaraldehyde fixation and proper quenching of unbound aldehyde groups minimizes chemical modification of the target, thus improv...

DNA replication is a basic biological process required for faithful copying and transmission of the genetic information during cell division. In higher eukaryotes, DNA replication is subjected to tight spatio-temporal regulation, which is manifested in sequential activation of replication origins1. Neighboring replication origins firing synchronously form clusters of replicons2. At the level of optical microscopy, sites of ongoing DNA replication are detected as replication foci of various number and size. Replication foci display specific patterns of spatial distribution within the cell nucleus depending on the replication timing of the labeled DNA3,4, which, in turn, is tightly correlated with its gene activity. Thanks to well-defined sequence of DNA replication, strictly ordered in space and time, replicative labeling is a powerful method of precise DNA labeling not only for the study of replication process per se, but also to discriminate a specific DNA sub-fraction with defined transcription activity and compaction level. Visualization of replicating chromatin is usually performed through the detection of major protein components of DNA replication machinery (either by immunostaining or by expression of fluorescent protein tags5,6) or by the incorporation of modified DNA synthesis precursors7,8,9,10. Of these, only methods based on the incorporation of modified nucleotides into newly replicated DNA allow for the capture of conformational changes in chromatin during replication, and trace the behavior of replicative domains after their replication is completed.
In higher eukaryotes, DNA packaging into chromatin adds another level of complexity to the regulation of basic genetic functions (transcription, replication, reparation, etc.). Chromatin folding affects accessibility of DNA to regulatory trans-factors and DNA conformational changes (double helix unwinding) required for the template synthesis. Therefore, it is generally accepted that DNA-dependent synthetic processes in the cell nucleus require a structural transition of chromatin from its condensed, repressive state to a more accessible, open conformation. Cytologically, these two chromatin states are defined as heterochromatin and euchromatin. However, there is still no consensus concerning the mode of DNA folding in the nucleus. The hypotheses range from a &#34;polymer melt&#34; model11, where nucleosomal fiber behaves as a random polymer for which the packing density is controlled by phase separation mechanisms, to hierarchical folding models postulating sequential formation of chromatin fiber-like structures of increasing thickness12,13. Hierarchical folding models recently gained support from molecular approaches based on the analysis of in situ DNA-DNA contacts (chromosome conformation capture, 3C), demonstrating the existence of the hierarchy of chromatin structural domains14. It is important to note that replication units correlate very well to these chromatin domains15. The major criticism of these models is based on potential artificial chromatin aggregation caused by sample preparation procedures, such as permeabilization of cell membranes and removal of non-chromatin components, in order to improve chromatin contrast for ultrastructural studies while improving chromatin accessibility for various probes (e.g., antibodies). Recent technical advances in selective DNA staining for electron microscopy by DNA-binding fluorophore-mediated photo-oxidation of diaminobenzidine (ChromEMT6) has allowed for the elimination of this obstacle. However, the same considerations hold true for electron microscopy visualization of replicating DNA17,18. Here we describe a technique that allows for the simultaneous high-resolution ultrastructural mapping of newly synthesized DNA and total chromatin in intact aldehyde-crosslinked cells. The technique combines detection of EdU-labeled DNA by Click-chemistry with biotinylated probes and streptavidin-Nanogold, and ChromEMT.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-ethynyl-2`-deoxyuridine (EdU)</td>
			<td>Thermo Fisher</td>
			<td>A10044</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(4-Morpholino)ethane Sulfonic Acid (MES)</td>
			<td>Fisher Scientific</td>
			<td>BP300-100</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 555-azide</td>
			<td>Termo Fisher</td>
			<td>A20012</td>
			<td></td>
		</tr>
		<tr>
			<td>biotin-azide</td>
			<td>Lumiprobe</td>
			<td>C3730</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine</td>
			<td>Boval</td>
			<td>LY-0080</td>
			<td></td>
		</tr>
		<tr>
			<td>DDSA</td>
			<td>SPI-CHEM</td>
			<td>26544-38-7</td>
			<td></td>
		</tr>
		<tr>
			<td>DMP-30</td>
			<td>SPI-CHEM</td>
			<td>90-72-2</td>
			<td></td>
		</tr>
		<tr>
			<td>DRAQ5</td>
			<td>Thermo Scientific</td>
			<td>62251</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy resin monomer</td>
			<td>SPI-CHEM</td>
			<td>90529-77-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (25%, EM Grade)</td>
			<td>TED PELLA, INC</td>
			<td>18426</td>
			<td></td>
		</tr>
		<tr>
			<td>Gum arabic</td>
			<td>ACROS Organics</td>
			<td>258850010</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Panreac</td>
			<td>141396.1209</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>SIGMA-ALDRICH</td>
			<td>213462</td>
			<td></td>
		</tr>
		<tr>
			<td>NMA</td>
			<td>SPI-CHEM</td>
			<td>25134-21-8</td>
			<td></td>
		</tr>
		<tr>
			<td>N-propyl gallate</td>
			<td>SIGMA-ALDRICH</td>
			<td>P3130</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>MP Biomedicals</td>
			<td>2810305</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver lactate</td>
			<td>ALDRICH</td>
			<td>359750-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-AlexaFluor 488 conjugate</td>
			<td>Termo Fisher</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-Nanogold conjugate</td>
			<td>Nanoprobes</td>
			<td>2016</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrachloroauric acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>CHEM-IMPEX INT&#39;L</td>
			<td>298</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fluka Chemica</td>
			<td>93420</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Coater</td>
			<td>Hitachi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Copper single slot grids</td>
			<td>Ted Pella</td>
			<td>1GC10H</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5 fluorescence filter set (Ex620/60 DM660 Em700/75)</td>
			<td>Nikon</td>
			<td>Cy5 HQ</td>
			<td>Alternatives: Zeiss, Leica, Olympus</td>
		</tr>
		<tr>
			<td>Diamond knife Ultra Wet 45o</td>
			<td>Diatome</td>
			<td>DU</td>
			<td>Alternatives: Ted Pella</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td>Alternatives: Zeiss, Leica, Olympus</td>
		</tr>
		<tr>
			<td>High-tilt sample holder</td>
			<td>Jeol</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator</td>
			<td>Biosan</td>
			<td>Multi Bio RS-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscope operating at 200 kV in EFTEM mode, with high-tilt goniometer</td>
			<td>Jeol</td>
			<td>JEM-2100</td>
			<td>Alternatives: FEI, Hitachi</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Ted Pella</td>
			<td>523</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramicrotome</td>
			<td>Leica</td>
			<td>UltraCut-E</td>
			<td>Alternatives: RMC</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition</td>
			<td>Open Source</td>
			<td>SerialEM (https://bio3d.colorado.edu/SerialEM/)</td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing</td>
			<td>Open Source</td>
			<td>IMOD (https://bio3d.colorado.edu/imod/)</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging replicative domains in ultrastructurally preserved chromatin by electron tomography

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, replication, segregation, etc.) remain poorly understood, partially due to the lack of experimental approaches to high-resolution visualization of specific chromatin loci in structurally preserved nuclei. Here we present a protocol for the visualization of replicative domains in monolayer cell culture in situ,&#160;by combining EdU labeling of newly synthesized DNA with subsequent label detection with Ag-amplification of Nanogold particles and ChromEM staining of chromatin. This protocol allows for the high-contrast, high-efficiency pre-embedding labeling, compatible with traditional glutaraldehyde fixation that provides the best structural preservation of chromatin for room-temperature sample processing. Another advantage of pre-embedding labeling is the possibility to pre-select cells of interest for sectioning. This is especially important for the analysis of heterogeneous cell populations, as well as compatibility with electron tomography approaches to high-resolution 3D analysis of chromatin organization at sites of replication, and the analysis of post-replicative chromatin rearrangement and sister chromatid segregation in the interphase.

Replication foci in mammalian cell nuclei display distinct patterns of distribution within the nucleus depending on S-phase progression. These patterns correlate with transcriptional activity of the loci being replicated. Since the method presented here utilizes a rather strongfixation procedure, it is fairly straightforward to use replicative pulse labeling for specific detection of chromatin loci in various transcriptional states, even under conditions offering best structural preservation of chromatin obtained by room...

Watch this Scientific Journal Video about Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography at JoVE.com

Imaging Replicative Domains in Ultrastructurally Preserved Chromatin by Electron Tomography

This protocol presents a technique for high-resolution mapping of replication sites in structurally preserved chromatin in situ that employs a combination of pre-embedding EdU-streptavidin-Nanogold labeling and ChromEMT.

Principles of DNA folding in the cell nucleus and its dynamic transformations that occur during the fulfillment of basic genetic functions (transcription, ...

We present a protocol for the electron microscopic visualization of the replicative domains in the cell by the EdU labeling of the newly synthesized DNA and label detection with silver enhancement of Nanogold particles and ChromEM staining of the chromatin. This protocol gives the high-contrast pre-embedding labeling with traditional glutaraldehyde fixation that gives the best structural preservation of the chromatin for the room temperature sample processing. This protocol is optimized for the adherence cell growing on the coverslips in the Petri dish.Add EdU to 10 micro moles final concentration and place the cells into the incubator for the desired labeling time. Fix the labeled cells with 2.5%gluaradehydrate in a 100 millimoles sodium cacodylate solution for one hour. Longer fixation time may adversely affect the EdU labeling.Remove glutaraldehyde by rinsing the samples with PBS supplemented with five millimoles of magnesium chloride, further regarded as PBS star. Wash three times for 10 minutes. Permeabilize plasma membranes with 1%solution of Trione X-100 in PBS star, make two changes for five minutes each.This solution is further referred to as PBS star T.Extensively wash the sample with PBS star. Five changes for five minutes each. Quench the residual aldehyde groups with 20 millimoles of glycine in PBS star, two times for 10 minutes.Place the sample in PBS star with 1%BSA for half an hour. While blocking in BSA, prepare the reaction cocktail for EdU detection. The order of component addition is important.EdU biotin-azide conjugation is performed in the moist chamber to minimize the evaporation and concentration shifts in the reaction cocktail. Prepare the moist chamber. Place the coverslip on the para-film with a cells facing up and layer 50 to 100 microliters of the reaction cocktail onto the coverslip.Reaction takes 30 minutes at room temperature. Stop the reaction by washing the sample in PBS star T, five times for five minutes each. Block again with 1%BSA in PBS star T for another half an hour.Prepare streptavidin-Nanogold solution in PBS star T with 1%BSA. Incubate the sample with streptavidin overnight at plus four degrees in a newly prepared moist chamber. The procedure is the same as with EdU biotin-azide conjugation.Stop the reaction and wash the sample, five times for 10 minutes each with PBS star T.Stabilize biotin streptavidin complex by additional fixation will 1%glutaraldehyde PBS star for half an hour. Remove glutaraldehyde by intense washing with deionized water. Quench residual aldehyde groups with one milligram per milliliter sodium borohydride.Wash again thoroughly. Next step is to increase the size of Nanogold particles by a means of silver deposition on the golden course. Prepare premixes to minimize darkroom activity.Equilibrate the samples with washing buffer and proceed to a darkroom. In the darkroom mix the components to prepare the washing solution and apply it to the samples. Acacia powder solution is highly visicles.So during mixing the components, rock the tube slowly to avoid bubble and foam formation. The reaction takes from two to five minutes. We recommend to perform a test run to determine the optimal reaction time.Longer incubation time may result in an unspecific silver deposition. To stop the reaction, quickly rinse the sample with three to five changes of the deionized water. Followed by three more changes for five minutes each.To protect the silver nanoparticles from dissolution by the osmium tetroxide, incubate the samples in 0.05%tetrachloroauric acid for two minutes in the dark. Wash thoroughly with deionized water. At this stage, additional contrast is added to the DNA containing material.Saturate the sample with DRAQ5 DNA stain. Add diamionobenzidine solution to the sample and incubate for one minute. Move the coverslip into the glass bottom Petri dish, and place it on the stage of the inverted microscope.Irradiate several fields of view with 640 nanometer red light. Left panel shows the complete photo bleaching of DRAQ5. On the right panel, the same field of view is imaged in the transmitted light showing DAB precipitate.Wash samples with deionized water. Next stage should be performed under the fume hood. Prepared partially reduced osmium tetroxide solution.Be careful. This substance is highly volatile and toxic. Apply the prepared solution onto the sample.At this stage, the reduced osmium compounds react with diamionobenzidine depositions and increase the contrast of the DNA. Dispose osmium containing solution according to your local regulation and wash samples three times for five minutes each. The sample is further dehydrated in a series of increasing ethanol concentrations followed by the infiltration of the cells with ethanol resin mixes.Replace alcohol resin mix with freshly prepared pure resin. Fill the properly sized embedding mold with a freshly prepared resin. Place the coverslip with a cells facing down over the resin filled cavity.Cure the resin for 24 hours at 37 degrees. Then at 60 degrees for at least two days. Remove resin from a bottom surface of a covers.Drop the slab into a liquid nitrogen and then transfer into boiling water. The glass should detach. Repeat if needed.Irradiated fields of view should be clearly visible due to the diamionobenzidine deposition and DAB staining. Cut out the area of interest from the slab using the hacksaw or any other suitable instrument. Place the cutout into the ultramicrotome simple holder.Set up the ultramicrotome for final block trimming. Using the razor blade, prepare the pyramid shaped pillet. The size of a flat area on top of the pyramid should be about 400 by 200 microns.Mount the prepared pyramid into the ultramicrotome arm and install the knife. Prepare the sections. Section thickness can be adjusted to suit the experimental requirements.We aim for EM tomography. So we prepared sections with a thickness of 250 nanometers also referred as semi-thin sections. Detach the sections from the knife's edge and mount them on a slot grid.We used a slot grid, they were round one millimeters to be around, one millimeter side opening. The small opening size provide better stability of the sections. Let the samples dry on the grid.At this moment, the sections are ready for observation. Place the grid into the electron microscope sample holder. Load the sample into the electron microscope.Acquire tilt series. Replication foci in mammalian cell nuclei display distinct patterns of distribution depending on the S-phase progression. Successful EdU labeling confirmed by the click reaction with fluorescently labeled azide demonstrates typical replication pattern in the heterogeneous cell population after glutaraldehyde fixation on top.Streptavidin labeling can be influenced by glutaraldehyde. So first check the fluorescence streptavidin staining under the same condition as streptavidin nanogold on the bottom. Good outcome of the silver enhancements procedure is a dark brown staining of some nuclei and very fainted yellow cytoplast stain, shown on the bottom.Advantage of a pre-embedding labeling is the possibility to select cells for the sectioning. It is possible at this stage because the labeling patterns become visible under a bright field microscope. The appropriate labeling results in the well demarcated replication sites.Arrowhead show the DAB stained chromatin fibers. The background labeling on the right is limited to a few nanoparticles per square micron. Semi-thin sections are ready for tomography and do not require addition of gold nanoparticles.The described approach used for the studies of chromatin organization at sites with enabled replication. For analysis of post replicative rearrangement of chromatin including high resolution imaging of chromatin segregation. It also used for replicated labeling of large chromosomal domains and studies of higher order chromatin folding and heterochromatin.This imaging technique is also important as a reference point for the data generated by other non microscopic approaches. The method can be also accommodated into correlated microscopic studies including super resolution techniques. This open new horizons in studies of chromatin higher order structure and its dynamics in the various physiological states of the cell.

Research

JoVE Journal

Biology

Chromatin Isolation by RNA Purification (ChIRP)

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