Name: high-resolution mapping of protein-dna interactions in mouse stem cell-derived neurons using chromatin immunoprecipitation-exonuclease (chip-exo)
Uploaded: 2020-08-14T16:00:00
Description: Watch this Scientific Journal Video about High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo at JoVE.com

We thank the member of the Rhee laboratory for sharing unpublished data and valuable discussions. This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) grant RGPIN-2018-06404 (H.R.).

NOTE: Autoclaved distilled and deionized water (ddH2O) is recommended for making buffers and reaction master mixes. Sections 1&#8722;4 describe cell lysis and sonication, sections 5&#8722;7 describe chromatin immunoprecipitation (ChIP), sections 8&#8722;11 describe enzymatic reactions on beads, sections 12 and 13 describe ChIP elution and DNA purification, and sections 14&#8722;19 describe library preparation.
1. Harvesting and crosslinking cells
<ol>
	<li>After d...

<ol>
	<li>Johnson, D. S., Mortazavi, A., Myers, R. M., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+in+vivo+protein-DNA+interactions.">Genome-wide mapping of in vivo protein-DNA interactions.</a> Science. 316 (5830), 1497-1502 (2007).</li><li>Patten, D. K., Corleone, G., Magnani, L., A, J. e. l. t. s. c. h., Rots, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenome+Editing:+Methods+and+Protocols.">Epigenome Editing: Methods and Protocols.</a> Methods in Molecular Biology. 1767, 271-288 (2018).</li><li>Serandour, A. A., Brown, G. D., Cohen, J. D., Carroll, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Illumina-based+ChIP-exonuclease+method+provides+insight+into+FoxA1-DNA+binding+properties.">Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties.</a> Genome Biology. 14 (12), 147 (2013).</li><li>Rhee, H. S., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+genome-wide+protein-DNA+interactions+detected+at+single-nucleotide+resolution.">Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution.</a> Cell. 147 (6), 1408-1419 (2011).</li><li>Rhee, H. S., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-exo+method+for+identifying+genomic+location+of+DNA-binding+proteins+with+near-single-nucleotide+accuracy.">ChIP-exo method for identifying genomic location of DNA-binding proteins with near-single-nucleotide accuracy.</a> Current Protocols in Molecular Biology. Chapter 21, 21-24 (2012).</li><li>Starick, S. R., 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=ChIP-exo+signal+associated+with+DNA-binding+motifs+provides+insight+into+the+genomic+binding+of+the+glucocorticoid+receptor+and+cooperating+transcription+factors.">ChIP-exo signal associated with DNA-binding motifs provides insight into the genomic binding of the glucocorticoid receptor and cooperating transcription factors.</a> Genome Research. 25 (6), 825-835 (2015).</li><li>Han, G. C., 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=Genome-Wide+Organization+of+GATA1+and+TAL1+Determined+at+High+Resolution.">Genome-Wide Organization of GATA1 and TAL1 Determined at High Resolution.</a> Molecular and Cellular Biology. 36 (1), 157-172 (2016).</li><li>Rhee, H. S., Bataille, A. R., Zhang, L. Y., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subnucleosomal+Structures+and+Nucleosome+Asymmetry+across+a+Genome.">Subnucleosomal Structures and Nucleosome Asymmetry across a Genome.</a> Cell. 159 (6), 1377-1388 (2014).</li><li>Rhee, H. S., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+structure+and+organization+of+eukaryotic+pre-initiation+complexes.">Genome-wide structure and organization of eukaryotic pre-initiation complexes.</a> Nature. 483 (7389), 295-301 (2012).</li><li>Zhou, X. F., Yan, Q., Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+regulon+of+a+GntR+family+regulator+via+transcriptome+and+ChIP-exo+analyses+and+its+contribution+to+virulence+in+Xanthomonas+citri.">Deciphering the regulon of a GntR family regulator via transcriptome and ChIP-exo analyses and its contribution to virulence in Xanthomonas citri.</a> Molecular Plant Pathology. 18 (2), 249-262 (2017).</li><li>Kim, 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=Systems+assessment+of+transcriptional+regulation+on+central+carbon+metabolism+by+Cra+and+CRP.">Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP.</a> Nucleic Acids Research. 46 (6), 2901-2917 (2018).</li><li>Niu, B., 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=In+vivo+genome-wide+binding+interactions+of+mouse+and+human+constitutive+androstane+receptors+reveal+novel+gene+targets.">In vivo genome-wide binding interactions of mouse and human constitutive androstane receptors reveal novel gene targets.</a> Nucleic Acids Research. 46 (16), 8385-8403 (2018).</li><li>Uuskula-Reimand, L., 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=Topoisomerase+II+beta+interacts+with+cohesin+and+CTCF+at+topological+domain+borders.">Topoisomerase II beta interacts with cohesin and CTCF at topological domain borders.</a> Genome Biology. 17, (2016).</li><li>Rhee, H. S., 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=Expression+of+Terminal+Effector+Genes+in+Mammalian+Neurons+Is+Maintained+by+a+Dynamic+Relay+of+Transient+Enhancers.">Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.</a> Neuron. 92 (6), 1252-1265 (2016).</li><li>Pugh, B. F., Venters, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Organization+of+Human+Transcription+Initiation+Complexes.">Genomic Organization of Human Transcription Initiation Complexes.</a> Plos One. 11 (2), (2016).</li><li>Wang, S., 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=ATF4+Gene+Network+Mediates+Cellular+Response+to+the+Anticancer+PAD+Inhibitor+YW3-56+in+Triple-Negative+Breast+Cancer+Cells.">ATF4 Gene Network Mediates Cellular Response to the Anticancer PAD Inhibitor YW3-56 in Triple-Negative Breast Cancer Cells.</a> Molecular Cancer Therapeutics. 14 (4), 877-888 (2015).</li><li>Barfeld, S. J., 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=c-Myc+Antagonises+the+Transcriptional+Activity+of+the+Androgen+Receptor+in+Prostate+Cancer+Affecting+Key+Gene+Networks.">c-Myc Antagonises the Transcriptional Activity of the Androgen Receptor in Prostate Cancer Affecting Key Gene Networks.</a> Ebiomedicine. 18, 83-93 (2017).</li><li>McHaourab, Z. F., Perreault, A. A., Venters, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq+and+ChIP-exo+profiling+of+Pol+II,+H2A.Z,+and+H3K4me3+in+human+K562+cells.">ChIP-seq and ChIP-exo profiling of Pol II, H2A.Z, and H3K4me3 in human K562 cells.</a> Scientific Data. 5, 180030 (2018).</li><li>Perreault, A. A., Sprunger, D. M., Venters, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+and+transcriptional+profiling+of+triple+negative+breast+cancer.">Epigenetic and transcriptional profiling of triple negative breast cancer.</a> Scientific Data. 6, 190033 (2019).</li><li>Rossi, M. J., Lai, W. K. M., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+ChIP-exo+assays.">Simplified ChIP-exo assays.</a> Nature Communications. 9 (1), 2842 (2018).</li><li>Perreault, A. A., Venters, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ChIP-exo+Method:+Identifying+Protein-DNA+Interactions+with+Near+Base+Pair+Precision.">The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision.</a> Journal of Visualized Experiments. (118), (2016).</li><li>Jezek, M., Jacques, A., Jaiswal, D., Green, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+Immunoprecipitation+(ChIP)+of+Histone+Modifications+from+Saccharomyces+cerevisiae.">Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae.</a> Journal of Visualized Experiments. (130), (2017).</li><li>Terranova, C., 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=An+Integrated+Platform+for+Genome-wide+Mapping+of+Chromatin+States+Using+High-throughput+ChIP-sequencing+in+Tumor+Tissues.">An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues.</a> Journal of Visualized Experiments. (134), (2018).</li><li>Pchelintsev, N. A., Adams, P. D., Nelson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Parameters+for+Efficient+Sonication+and+Improved+Chromatin+Immunoprecipitation+of+High+Molecular+Weight+Proteins.">Critical Parameters for Efficient Sonication and Improved Chromatin Immunoprecipitation of High Molecular Weight Proteins.</a> PLos One. 11 (1), (2016).</li><li>Yamada, N., Lai, W. K. M., Farrell, N., Pugh, B. F., Mahony, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+protein-DNA+binding+event+subtypes+in+ChIP-exo+data.">Characterizing protein-DNA binding event subtypes in ChIP-exo data.</a> Bioinformatics. 35 (6), 903-913 (2019).</li><li>Yen, K., Vinayachandran, V., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SWR-C+and+INO80+chromatin+remodelers+recognize+nucleosome-free+regions+near++1+nucleosomes.">SWR-C and INO80 chromatin remodelers recognize nucleosome-free regions near +1 nucleosomes.</a> Cell. 154 (6), 1246-1256 (2013).</li><li>Vinayachandran, V., 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=Widespread+and+precise+reprogramming+of+yeast+protein-genome+interactions+in+response+to+heat+shock.">Widespread and precise reprogramming of yeast protein-genome interactions in response to heat shock.</a> Genome Research. , (2018).</li></ol>

In this protocol, ChIP followed by exonuclease digestion is used to obtain DNA libraries for the identification of protein-DNA interactions in mammalian cells at ultra-high mapping resolution. Many variables contribute to the quality of the ChIP-exo experiment. Critical experimental parameters include the quality of antibodies, optimization of sonication, and the number of LM-PCR cycles. These critical experimental parameters are also what can limit ChIP-exo experiments and will be&#160;discussed below.
<p class="jov...

The locations of protein-DNA interactions provide insight into the mechanisms of gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) has been used for a decade to examine genome-wide protein-DNA interactions in living cells1,2. However, the ChIP-seq method is limited by heterogeneity in DNA fragmentation and unbound DNA contamination that lead to low mapping resolution, false positives, missed calls, and non-specific background signal. The combination of ChIP with exonuclease digestion (ChIP-exo) improves upon the ChIP-seq method by trimming ChIP DNA to the protein-DNA crosslinking points, providing near base-pair resolution and a low background signal3,4,5. The greatly improved mapping resolution and low background provided by ChIP-exo allow accurate and comprehensive protein-DNA binding locations to be determined across the genome. ChIP-exo is able to reveal functionally distinct DNA-binding motifs, cooperative interactions between transcription factors (TF), and multiple protein-DNA crosslinking sites in a certain genomic binding location,&#160;not detectable by other genomic mapping methods3,4,6,7.
ChIP-exo was initially used in budding yeast to examine the sequence-specific DNA binding of TFs, to study the precise organization of the transcription pre-initiation complex, and sub-nucleosomal structure of individual histones across the genome4,8,9. Since its introduction in 20114, ChIP-exo has been successfully utilized in many other organisms including bacteria, mice, and human cells7,10,11,12,13,14,15,16,17. In 2016, Rhee et al.14 used ChIP-exo in mammalian neurons for the first time to understand how neuronal gene expression was maintained after the downregulation of programming TF Lhx3, which forms a heterodimer complex with another programming TF&#160;Isl1. This&#160;study showed that in the absence of Lhx3, Isl1 is recruited to new neuronal enhancers bound by Onecut1 TF to maintain gene expression of neuronal effector genes. In this study, ChIP-exo revealed how multiple TFs dynamically recognize cell type&#160;and cell stage-specific DNA regulatory elements in a combinatorial manner at near-nucleotide mapping resolution. Other studies also used the ChIP-exo method to understand the interplay between proteins and DNA in other mammalian cell lines. Han et al.7 used ChIP-exo to examine genome-wide organization of GATA1 and TAL1 TFs in mouse erythroid cells using ChIP-exo. This study found that TAL1 is directly recruited to DNA rather than indirectly through protein-protein interactions with GATA1 throughout erythroid differentiation. Recent studies also used ChIP-exo to profile the genome-wide binding locations of CTCF, RNA Polymerase II, and histone marks to study epigenomic and transcriptional mechanisms in human cell lines18,19.
There are several versions of the ChIP-exo protocol available3,5,20. However, these ChIP-exo protocols are difficult to follow for researches who are not familiar with next-generation sequencing library preparation. An excellent version of the ChIP-exo protocol was published with easy-to-follow instructions and a video21, but contained&#160;many enzymatic steps that require a significant amount of time to complete. Here we report a new version of the ChIP-exo protocol containing reduced&#160;enzymatic steps and&#160;incubation times, and explanations for each enzymatic step21. End repair and dA-tailing reactions are combined in a single step using end prep enzyme. The incubation times for index and universal adapter ligation steps are reduced from 2 h to 15 min using a ligation enhancer. The kinase reaction after the index adapter ligation step described in the previous ChIP-exo protocol is removed. Instead, a phosphate group is added during oligo DNA synthesis to one of the 5&#8217; ends of the index adapter (Table 1), which will be used for the lambda exonuclease digestion step. While the previous ChIP-exo protocol used&#160;RecJf exonuclease digestion to eliminate single-stranded DNA contaminants, this digestion step is removed here because it is not critical for the quality of the ChIP-exo library. In addition, to purify reverse-crosslinked DNA after ChIP elution, a magnetic beads purification method is used instead of the phenol:chloroform:isoamyl alcohol (PCIA) extraction method. This reduces the incubation time of DNA extraction. Importantly, it removes the majority of adapter dimers formed during index adapter ligation, which may impact the efficiency of ligation-mediated PCR.
The ChIP-exo protocol presented here is optimized for the detection of precise protein-DNA interactions in mammalian neurons differentiated from mouse embryonic stem (ES) cells. Briefly, harvested and crosslinked neuronal cells are lysed, to allow&#160;chromatin to be exposed to sonication, then sonicated so that appropriately sized DNA fragments are obtained (Figure 1). Antibody-coated beads are then used to selectively immunoprecipitate fragmented, soluble chromatin to the protein of interest. While the immunoprecipitated DNA is still on the beads, end-repair, ligation of sequencing adapters, fill-in reaction&#160;and 5&#8217; to 3&#8217; lambda exonuclease digestion steps are performed. The exonuclease digestion step is what gives ChIP-exo its ultra-high resolution and high signal-to-noise ratio. Lambda exonuclease trims the immunoprecipitated DNA a few base-pairs (bp) from the crosslinking site, thus causing contaminating DNA to be degraded. The exonuclease-treated ChIP DNA is eluted from the antibody-coated beads, protein-DNA crosslinks are reversed, and proteins are degraded. DNA is extracted and denatured to single-stranded ChIP DNA, followed by primer annealing and extension to make double-stranded DNA (dsDNA). Next, ligation of a universal adapter to the exonuclease-treated ends is performed. The resulting DNA is purified, then PCR amplified, gel purified, and subjected to next-generation sequencing.
The ChIP-exo protocol is longer than the ChIP-seq protocol, but is not very technically challenging. Any successfully immunoprecipitated ChIP DNA can be subjected to ChIP-exo, with several additional enzymatic steps. The notable advantages of ChIP-exo, such as ultra-high mapping resolution, a reduced background signal, and decreased false positive&#160;and negatives, regarding genomic binding sites, outweigh the time cost.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Agarose, UltraPure</td>
			<td >Invitrogen</td>
			<td >16500</td>
			<td >Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1)</td>
		</tr>
		<tr >
			<td >Albumin, Bovine Serum (BSA), Protease Free, Heat Shock Isolation, Min. 98%</td>
			<td >BioShop</td>
			<td >ALB003</td>
			<td >Blocking Solution</td>
		</tr>
		<tr >
			<td >Antibody against Isl1</td>
			<td >DSHB</td>
			<td >39.3F7</td>
			<td >Antibody incuation with beads (Section 5.6)</td>
		</tr>
		<tr >
			<td >Bioruptor Pico&#160;</td>
			<td >Diagenode</td>
			<td >B01060010</td>
			<td >Sonicating Chromatin (Section 3.3)</td>
		</tr>
		<tr >
			<td >Bovine serum albumin (BSA), Molecular Biology Grade</td>
			<td >New England BioLabs</td>
			<td >B9000S</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2)</td>
		</tr>
		<tr >
			<td >Centrifuge 5424 R</td>
			<td >Eppendorf</td>
			<td >5404000138</td>
			<td >Sonicating Chromatin (Section 3.5), Checking Sonication (Section 4.3.1, 4.3.3 and 4.3.4)</td>
		</tr>
		<tr >
			<td >Centrifuge 5804 R</td>
			<td >Eppendorf</td>
			<td >22623508</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.3), Cell lysis (Section 2.2 and 2.3), Sonicating Chromatin (Section 3.1)</td>
		</tr>
		<tr >
			<td >cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td >Roche</td>
			<td >4693159001</td>
			<td >Added to all buffers, except Proteinase K buffer and ChIP Elution buffer</td>
		</tr>
		<tr >
			<td >dATP Solution</td>
			<td >New England BioLabs</td>
			<td >N0440S</td>
			<td >dA-Tailing Reaction (Section 15.1)</td>
		</tr>
		<tr >
			<td >Deoxycholic Acid Sodium Salt</td>
			<td >fisher scientific</td>
			<td >BP349</td>
			<td >Lysis Buffer 3, High Salt Wash Buffer and LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >dNTP Mix, Molecular Biology Grade</td>
			<td >Thermo Scientific</td>
			<td >R0192</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2)</td>
		</tr>
		<tr >
			<td >DreamTaq Green PCR Master Mix, 2x&#160;</td>
			<td >Thermo Scientific</td>
			<td >K1081</td>
			<td >Ligation-Mediated PCR (Section 18.1)</td>
		</tr>
		<tr >
			<td >EDTA, 0.5 M, Sterile Solution, pH 8.0</td>
			<td >BioShop</td>
			<td >EDT111</td>
			<td >Lysis Buffer 1-3, Checking Sonication (Section 4.1), High Salt Wash Buffer, LiCl Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Ethyl Alcohol Anhydrous, 100%</td>
			<td >Commercial alcohols</td>
			<td >P006EAAN</td>
			<td >Checking Sonication (Section 4.3.3)</td>
		</tr>
		<tr >
			<td >Formaldehyde, 36.5-38%, contains 10-15% methanol</td>
			<td >Sigma</td>
			<td >F8775</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.1)</td>
		</tr>
		<tr >
			<td >Glycerol, Reagent Grade, min 99.5%</td>
			<td >BioShop</td>
			<td >GLY002</td>
			<td >Lysis Buffer 1</td>
		</tr>
		<tr >
			<td >Glycine, Biotechnology Grade, min. 99%</td>
			<td >BioShop</td>
			<td >GLN001</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.2)</td>
		</tr>
		<tr >
			<td >Glycogen, RNA Grade</td>
			<td >Thermo Scientific</td>
			<td >R0551</td>
			<td >Checking Sonication (Section 4.3.3)</td>
		</tr>
		<tr >
			<td >HEPES, 1 M Sterile-filtered Solution, pH 7.3&#160;</td>
			<td >BioShop</td>
			<td >HEP003</td>
			<td >Lysis Buffer 1, High Salt Wash Buffer</td>
		</tr>
		<tr >
			<td >Klenow Fragment (3-&#62;5 exo-)</td>
			<td >New England BioLabs</td>
			<td >M0212S</td>
			<td >dA-Tailing Reaction (Section 15.1)</td>
		</tr>
		<tr >
			<td >Lambda exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0262S</td>
			<td >Lambda Exonuclease Digestion on Beads (Section 11.2)</td>
		</tr>
		<tr >
			<td >Ligase Enhancer</td>
			<td >New England BioLabs</td>
			<td >E7645S</td>
			<td >NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1)</td>
		</tr>
		<tr >
			<td >Ligase Master Mix</td>
			<td >New England BioLabs</td>
			<td >E7645S</td>
			<td >NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1)</td>
		</tr>
		<tr >
			<td >Lithium Chloride (LiCl), Reagent grade</td>
			<td >Bioshop</td>
			<td >LIT704</td>
			<td >LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >Magnetic beads for ChIP (Dynabeads Protein G)</td>
			<td >Dynabeads Protein G (magnetic beads for ChIP)</td>
			<td >Dynabeads Protein G (magnetic beads for ChIP)</td>
			<td >Antibody incubation with beads (Section 5)</td>
		</tr>
		<tr >
			<td >Magnetic beads for DNA purification (AMPure XP Beads)</td>
			<td >Beckman Coulter</td>
			<td >A63880</td>
			<td >DNA Extraction (Section 13.3) and DNA Clean-up (Section 17.1)</td>
		</tr>
		<tr >
			<td >Magnetic rack (DynaMag-2 Magnet)</td>
			<td >Invitrogen</td>
			<td >12321D</td>
			<td >Used in many steps in Sections: 5 - 11, 13</td>
		</tr>
		<tr >
			<td >MinElute Gel Extraction Kit</td>
			<td >Qiagen&#160;</td>
			<td >28604</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.2)</td>
		</tr>
		<tr >
			<td >N-Lauroylsarcosine sodium salt solution, 30% aqueous solution, &#8805;97.0% (HPLC)</td>
			<td >Sigma</td>
			<td >61747</td>
			<td >Lysis Buffer 3</td>
		</tr>
		<tr >
			<td >Octylphenol Ethoxylate (IGEPAL CA630)</td>
			<td >BioShop</td>
			<td >NON999</td>
			<td >Lysis Buffer 1 and LiCl Wash Buffer</td>
		</tr>
		<tr >
			<td >Phenol:Chloroform:Isoamyl Alcohol, Biotechnology Grade (25:24:1)</td>
			<td >BioShop</td>
			<td >PHE512</td>
			<td >Checking Sonication (Section 4.3.1)</td>
		</tr>
		<tr >
			<td >phi29 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0269L</td>
			<td >Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.3 and 14.4)</td>
		</tr>
		<tr >
			<td >Phosphate-Buffered Saline (PBS), 1x&#160;</td>
			<td >Corning</td>
			<td >21040CV</td>
			<td >Harvest, cross-linking and freezing cells (Section 1.3) and Sonicating Chromatin (Section 3.1), Antibody incuation with beads (Section 5.1)</td>
		</tr>
		<tr >
			<td >PowerPac Basic Power Supply</td>
			<td >BioRad</td>
			<td >1645050</td>
			<td >Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1)</td>
		</tr>
		<tr >
			<td >ProFlex PCR System</td>
			<td >Applied Biosystems</td>
			<td >ProFlex PCR System</td>
			<td >Used in Sections: 14.2, 14.4, 15.2, 16.2 and 18.2</td>
		</tr>
		<tr >
			<td >Protein LoBind Tube, 2.0 mL&#160;</td>
			<td >Eppendorf</td>
			<td >22431102</td>
			<td >Antibody Incubation with Beads (Section 5.2) and Chromatin Immunoprecipitation (Section 6.3)</td>
		</tr>
		<tr >
			<td >Proteinase K Solution, RNA Grade</td>
			<td >Invitrogen</td>
			<td >25530049</td>
			<td >Checking Sonication (Section 4.2) and Elution and Reverse Crosslinking (Section 12.3)</td>
		</tr>
		<tr >
			<td >Qubit 4.0 Fluorometer</td>
			<td >Invitrogen</td>
			<td >Q33226</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.3)</td>
		</tr>
		<tr >
			<td >Quibit dsDNA BR assay kit</td>
			<td >Invitrogen</td>
			<td >Q32853</td>
			<td >Gel Purification of PCR Amplified DNA (Section 19.3)</td>
		</tr>
		<tr >
			<td >Rnase A, Dnase and Protease-free</td>
			<td >Thermo Scientific</td>
			<td >EN0531</td>
			<td >Checking Sonication (Section 4.3.2) and DNA Extraction (Section 13.2)</td>
		</tr>
		<tr >
			<td >Sodium chloride (NaCl), BioReagent&#160;</td>
			<td >Sigma</td>
			<td >S5886</td>
			<td >Lysis Buffer 1-3, High Salt Wash Buffer</td>
		</tr>
		<tr >
			<td >Sodium Dodecyl Sulfate (SDS), Electrophoresis Grade</td>
			<td >BioShop</td>
			<td >SDS001</td>
			<td >Checking Sonication (Section 4.1), High Salt Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Sonication beads and 15 mL Bioruptor Tubes</td>
			<td >Diagenode</td>
			<td >C01020031</td>
			<td >Sonicating Chromatin (Section 3.1 and 3.2)</td>
		</tr>
		<tr >
			<td >ThermoMixer F1.5&#160;</td>
			<td >Eppendorf</td>
			<td >5384000020</td>
			<td >Section 4.2, 4.3.2, 4.3.5, 8.2, 9.2, 10.3, 11.2, 12.2, 12.3, 13.2 and 16.2</td>
		</tr>
		<tr >
			<td >Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 7.4</td>
			<td >Sigma</td>
			<td >T2194</td>
			<td >10 mM Tris-HCl Buffer</td>
		</tr>
		<tr >
			<td >Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 8.0</td>
			<td >Sigma</td>
			<td >T2694</td>
			<td >Lysis Buffer 2, Lysis Buffer 3, Checking Sonication (Section 4.1), LiCl Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr >
			<td >Ultra II End Repair/dA-Tailing Module (24 rxn -&#62; 120 rxn)</td>
			<td >New England BioLabs</td>
			<td >E7546S</td>
			<td >End Prep Reaction mix and End Prep Enzyme mix. End-repair and dA-Tailing Reaction on Beads (Section 8.2)</td>
		</tr>
		<tr >
			<td >VWR Mini Tube Rocker, Variable Speed</td>
			<td >VWR</td>
			<td >10159-752</td>
			<td >Used in many steps in sections: 1, 2, 5, 6 and 7</td>
		</tr>
		<tr >
			<td >2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Triton X-100, Reagent Grade</td>
			<td >BioShop</td>
			<td >TRX506</td>
			<td >Lysis Buffer 1, Sonicating Chromatin (Section 3.4) and High Salt Wash Buffer</td>
		</tr>
	</tbody>
</table>

high-resolution mapping of protein-dna interactions in mouse stem cell-derived neurons using chromatin immunoprecipitation-exonuclease (chip-exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) is widely used to identify genome-wide binding locations of DNA-binding proteins. However, the ChIP-seq method is limited by its heterogeneity in length of sonicated DNA fragments and non-specific background DNA, resulting in low mapping resolution and uncertainty in DNA-binding sites. To overcome these limitations, the combination of ChIP with exonuclease digestion (ChIP-exo) utilizes 5&#8217; to 3&#8217; exonuclease digestion to trim the heterogeneously sized immunoprecipitated DNA to the protein-DNA crosslinking site. Exonuclease treatment also eliminates non-specific background DNA. The library-prepared and exonuclease-digested DNA can be sent for high-throughput sequencing. The ChIP-exo method allows for near base-pair mapping resolution with greater detection sensitivity and reduced background signal. An optimized ChIP-exo protocol for mammalian cells and next-generation sequencing is described below.

Figure 2A&#160;illustrates&#160;sonication results after cell lysis and sonication, with various cycles, of motor neuron cells differentiated from mouse ES cells. The optimal number of sonication cycles (for example, 12 cycles in Figure 2A) generated strong DNA intensity in 100&#8722;400 bp DNA fragments. High-quality ChIP-exo libraries are based on the size and quantity of fragmented chromatin DNA. Thus, optimization of sonication is recommended for each cell t...

Watch this Scientific Journal Video about High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo at JoVE.com

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease ChIP-Exo

Precise determination of protein-binding locations across the genome is important for understanding gene regulation. Here we describe a&#160;genomic mapping method that treats chromatin-immunoprecipitated DNA with exonuclease digestion (ChIP-exo) followed by high-throughput sequencing. This method detects protein-DNA interactions with near base-pair mapping resolution and high signal-to-noise ratio in mammalian neurons.

High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo)

Identification of specific protein-DNA interactions on the genome is important for understanding gene regulation. Chromatin immunoprecipitation coupled ...

The main advantage of our ChIP-exo method is the improved mapping resolution of protein DNA interactions in the cell compared to the conventional chromatin immunoprecipitation method. To prepare protein G magnetic beads for ChIP, mix the magnetic beads until homogeneous. Then add 25 microliters of the magnetic beads to a two milliliter protein low bind tube.Add one milliliter of blocking solution to the beads. Mix well by pipetting, then place the tube on a magnetic rack for one minute. Once the supernatant is clear, remove it.Next, add one milliliter of blocking solution to the beads. Place the tube on a rocking platform at four degrees Celsius and rock for 10 minutes, then briefly spin the tube. Place the tube on the magnetic rack and remove the supernatant.Add 500 microliters of blocking solution to the magnetic beads. Briefly spin the antibody against Islet-1, then add four micrograms of the antibody to the tube containing the magnetic beads. Place the tube containing magnetic beads and Islet-1 antibody on a rocking platform at four degrees Celsius and rock for six to 24 hours.To begin the chromatin immunoprecipitation, wash the antibody-coated beads in one milliliter of blocking solution. Place the tube containing the antibody-coated beads on a rocking platform at four degrees Celsius for five minutes. After briefly spinning, place the tube on a magnetic rack and remove the supernatant.Resuspend the antibody-coated beads in 50 microliters of blocking solution. Add one milliliter of sonicated lysates to the antibody-coated beads, then incubate the sample on a rocking platform at four degrees Celsius overnight. After allowing the sample to incubate overnight, collect liquid from the cap by briefly spinning the tube, then place the tube on a magnetic rack.And after one minute, remove the supernatant carefully with a pipette. Next, perform a series of washes as follows. Add one milliliter of cold wash buffer containing CPI to the sample.Mix the sample on a rocking platform at four degrees Celsius for five minutes. Briefly spin the sample tube, then place on a magnetic rack. After one minute, remove the supernatant with a pipette.For the first enzymatic reaction and repair and DA tailing, add 38 microliters of autoclaved double distilled water to the sample, then add end prep reaction mix and end prep enzyme mix. Mix the contents of the tube by pipetting, then incubate the sample at 20 degrees Celsius for 30 minutes. Next, wash the sample beads with high salt wash buffer, lithium chloride wash buffer, and 10 millimolar Tris hydrogen chloride buffer as described previously.To perform the index adapter ligation, add 27 microliters of cold 10 millimolar Tris hydrogen chloride buffer to the sample. Then add index adapter, ligation enhancer, and ligase master mix. Incubate the sample at 20 degrees Celsius for 15 minutes.Again, wash the sample beads with high salt wash buffer, lithium chloride wash buffer, and 10 millimolar Tris hydrogen chloride buffer. Then add 47 microliters of cold 10 millimolar Tris hydrogen chloride buffer to the sample. To repair the nick in the DNA resulting from the missing phosphodiester bond, add 11.1 microliters of the fill-in mix to the sample.Incubate the sample at 30 degrees Celsius for 20 minutes. After a series of washes, add 50 microliters of cold autoclaved double distilled water to the sample. To digest the ChIP DNA in the five prime to three prime direction, add lambda exonuclease buffer and lambda exonuclease and mix the sample by pipetting.Incubate the sample at 37 degrees Celsius for 30 minutes. After the digestion is complete, repeat the three washes a final time. To elute ChIP sample from the beads, resuspend the sample in 75 microliters of ChIP elution buffer and incubator at 65 degrees Celsius for 15 minutes at 130 times G.Add 2.5 microliters of 20 milligrams per milliliter proteinase K to the sample.Vortex the sample briefly, then incubate overnight at 65 degrees Celsius. Briefly spin the sample and place on a magnetic rack. After one minute, transfer the supernatant to a new 1.5 milliliter tube.Purify and elute the DNA. After purifying the eluted DNA, transfer 16 microliters of the extracted DNA sample to a PCR tube. Add 1.2 microliters of denaturing and primer annealing mix to the sample.Using the program described in table six of the manuscript, denature and anneal primers to the template DNA. Add three microliters of primer extension mix to the sample and run the PCR program described in table seven of the manuscript. Add 4.1 microliters of DA tailing mix to the sample and run the sample using the PCR program in table eight of the manuscript.Then add 21.5 microliters of universal adapter ligation mix to the sample and incubate for 15 minutes at 20 degrees Celsius. Add 29 microliters of LM-PCR mix to the sample, then run the sample using the program from table 10 of the manuscript. Following 18 cycles of LM-PCR, ChIP-exo samples from mouse motor neurons were electrophoresed on a 1.5%agarose gel.The results for ChIP-exo Islet-1 showed amplified DNA libraries around 200 to 400 base pairs indicating that the ChIP-exo libraries were successfully amplified by LM-PCR. The band around 100 base pairs represents artifacts from adapters and PCR primers. The no antibody control demonstrates that non-specific background DNA was digested by the lambda exonuclease treatment.Using ChIP-exo and ChIP-seq, Islet-1 bound locations were identified. The ChIP-exo signal was highly focused at Islet-1 binding sites detecting multiple clustered Islet-1 transcription factor binding patterns. The ChIP-seq signal displayed broader signals indicating that ChIP-exo had higher mapping resolution than ChIP-seq for Islet-1.Multiple transcription factors often bind DNA next to each other as a protein-DNA complex. ChIP-exo allows us to study how multiple transcription factors recognize and regulate target DNA.

high-resolution-mapping-protein-dna-interactions-mouse-stem-cell

Research

JoVE Journal

Genetics

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...