Name: the chip-exo method: identifying protein-dna interactions with near base pair precision
Uploaded: 2016-12-23T15:00:00
Description: Watch this Scientific Journal Video about The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision at JoVE.com

We thank the Venters Lab and members of the Molecular Physiology and Biophysics Department for helpful discussions. Special thanks to Frank Pugh for his guidance, mentoring, and many insightful discussions on the nuances of ChIP-exo while I was a post-doctoral fellow in his laboratory.

Note: Double distilled H2O or molecular grade equivalent is recommended for all buffers and reactions mixes.
Day 0: Material Preparation and Cell Harvest
1. Buffer Preparation
<ol>
	<li>Prepare Lysis Buffers 1 - 3 (Tables 1 - 3) and add 100 &#181;l complete protease inhibitor stock (CPI) to 50 ml of each buffer just prior to use. Prepare CPI stock by dissolving one tablet in 1 ml molecular grade H2O.</li>
	<li>Prepare ChIP Buff...

<ol>
	<li>Venters, B. J., 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=How+eukaryotic+genes+are+transcribed.">How eukaryotic genes are transcribed.</a> Crit Rev Biochem Mol Biol. 44, 117-141 (2009).</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, 1408-1419 (2011).</li><li>Chen, 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=Single-molecule+dynamics+of+enhanceosome+assembly+in+embryonic+stem+cells.">Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.</a> Cell. 156, 1274-1285 (2014).</li><li>Wales, S., Hashemi, S., Blais, A., McDermott, J. 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W., 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+ancient+protein-DNA+interaction+underlying+metazoan+sex+determination.">An ancient protein-DNA interaction underlying metazoan sex determination.</a> Nat Struct Mol Biol. 22, 442-451 (2015).</li><li>Zere, T. 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=Genomic+Targets+and+Features+of+BarA-UvrY+(-SirA)+Signal+Transduction+Systems.">Genomic Targets and Features of BarA-UvrY (-SirA) Signal Transduction Systems.</a> PLoS One. 10, e0145035 (2015).</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, 295-301 (2012).</li><li>Rhee, H. S., Bataille, A. R., Zhang, L., 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. , 1377-1388 (2014).</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, e0149339 (2016).</li><li>Albert, I., Wachi, S., Jiang, C., 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=GeneTrack--a+genomic+data+processing+and+visualization+framework.">GeneTrack--a genomic data processing and visualization framework.</a> Bioinformatics. 24, 1305-1306 (2008).</li><li>Guo, Y., Mahony, S., Gifford, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+genome+wide+binding+event+finding+and+motif+discovery+reveals+transcription+factor+spatial+binding+constraints.">High resolution genome wide binding event finding and motif discovery reveals transcription factor spatial binding constraints.</a> PLoS computational biology. 8, e1002638 (2012).</li><li>Bardet, A. F., 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=Identification+of+transcription+factor+binding+sites+from+ChIP-seq+data+at+high+resolution.">Identification of transcription factor binding sites from ChIP-seq data at high resolution.</a> Bioinformatics. 29, 2705-2713 (2013).</li><li>Wang, 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=MACE:+model+based+analysis+of+ChIP-exo.">MACE: model based analysis of ChIP-exo.</a> Nucleic Acids Res. 42, e156 (2014).</li><li>Bentley, D. 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=Accurate+whole+human+genome+sequencing+using+reversible+terminator+chemistry.">Accurate whole human genome sequencing using reversible terminator chemistry.</a> Nature. 456, 53-59 (2008).</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> Curr Protoc Mol Biol. Chapter 21, Unit 21.24 (2012).</li><li>Landt, S. G., 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-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia.">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Res. 22, 1813-1831 (2012).</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 Biol. 14, R147 (2013).</li><li>He, Q., Johnston, J., Zeitlinger, 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-nexus+enables+improved+detection+of+in+vivo+transcription+factor+binding+footprints.">ChIP-nexus enables improved detection of in vivo transcription factor binding footprints.</a> Nat Biotechnol. 33, 395-401 (2015).</li></ol>

We present a functional genomic protocol to determine the precise binding location for chromatin interacting proteins in an unbiased, genome-wide manner at near base pair resolution. The most critical step to achieve near base pair mapping resolution is the exonuclease treatment of the ChIP-enriched DNA while the immunoprecipitate remains on the magnetic resin. Ostensibly, protein complexes could potentially block in vivo footprinting of any given subunit (e.g., chromatin remodeling complexes or the nuc...

Chromatin immunoprecipitation (ChIP) is a powerful method to study mechanisms of gene regulation by selectively enriching for DNA fragments that interact with a given protein in living cells. Detection methods of ChIP-enriched DNA fragments have evolved as technology improves, from detection of a single locus (standard ChIP-qPCR) to hybridization on oligonucleotide microarrays (ChIP-chip) to high-throughput sequencing (ChIP-seq) 1. Although ChIP-seq has seen widespread application, chromatin heterogeneity and nonspecific DNA interactions have hampered data quality leading to false positives and imprecise mapping. To circumvent these limitations, Dr. Frank Pugh developed the ChIP-exo method 2. The salient feature of ChIP-exo is that it incorporates a 5&#39; to 3&#39; exonuclease, effectively footprinting transcription factor binding locations. As a result, the ChIP-exo methodology achieves higher resolution, greater dynamic range of detection, and lower background noise.
Although ChIP-exo is more technically challenging to master than ChIP-seq, it is being widely adopted as studies aim to gain unique ultra high-resolution insights using diverse biological systems 3-8. Indeed, ChIP-exo has been successfully applied to bacteria, yeast, mouse, rat, and human cell systems. As proof of principle, ChIP-exo was originally used to identify the precise binding motif for a handful of yeast transcription factors 2. The technique was also used in yeast to study the organization of the transcription pre-initiation complex, and to decipher subnucleosomal structure of various histones 9,10. More recently, we leveraged ChIP-exo to resolve adjacent TFIIB and Pol II binding events at human promoters, and showed that widespread divergent transcription arises from distinct initiation complexes 11.
The workflow presented here is optimized and streamlined for mammalian ChIP-exo (Figure 1). First, living primary or tissue culture cells are treated with formaldehyde to preserve in vivo protein-DNA interactions through a covalent crosslink. The cells are lysed and chromatin sheared to ~ 100 - 500 base pair size fragments. ChIP then selectively enriches for DNA fragments crosslinked to the protein of interest. At this point, ChIP-seq libraries are typically prepared, which inherently limits the detection resolution to the average fragment size of a few hundred base pairs. However, ChIP-exo overcomes this limitation by trimming the left and right 5&#39; DNA borders of the protein-DNA crosslink site with lambda exonuclease. Sequencing libraries are then constructed from exonuclease digested DNA as detailed below. The resulting nested 5&#39; borders represent an in vivo footprint of the protein-DNA interaction (Figure 1, step 14), and are detected by high throughput sequencing. Although the ChIP-exo methodology is more involved than ChIP-seq, transitions between most steps requires simple bead washing, which minimizes sample loss and experimental variability. Importantly, since ChIP-exo is a refined version of ChIP-seq, any sample that is successful with ChIP-seq should also be successful with ChIP-exo.
The in vivo footprinting of protein-DNA interactions with ChIP-exo results in a fundamentally distinct data structure from ChIP-seq. Although common ChIP-seq callers may be applied to ChIP-exo data, to obtain the most precise peak calls we recommend bioinformatics tools specifically designed with the unique structure of ChIP-exo data in mind. These include Genetrack, GEM, MACE, Peakzilla, and ExoProfiler 12-15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >37% formaldehyde, methanol free, 10x10ml ampules</td>
			<td >ThermoFisher Scientific</td>
			<td >28908</td>
			<td >Section 3</td>
		</tr>
		<tr >
			<td >Complete Protease Inhibitor cocktail (CPI)</td>
			<td >Roche Life Science</td>
			<td >11873580001</td>
			<td >Sections 4, 8</td>
		</tr>
		<tr >
			<td >Bioruptor sonicator</td>
			<td >Diagenode</td>
			<td >UCD200</td>
			<td >Section 5</td>
		</tr>
		<tr >
			<td >15 ml polystyrene tubes</td>
			<td >BD Falcon</td>
			<td >352095</td>
			<td >Section 5</td>
		</tr>
		<tr >
			<td >MagSepharose Protein G Xtra beads</td>
			<td >GE Healthcare</td>
			<td >28-9670-66</td>
			<td >Section 6</td>
		</tr>
		<tr >
			<td >DynaMag-1.5ml Side Magnetic Rack</td>
			<td >Invitrogen</td>
			<td >12321D</td>
			<td >Sections 6-17, 22</td>
		</tr>
		<tr >
			<td >Mini-Tube Rotator</td>
			<td >Fisher Scientific</td>
			<td >05-450-127</td>
			<td >Sections 7, 22</td>
		</tr>
		<tr >
			<td >T4 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0203</td>
			<td >Section 9</td>
		</tr>
		<tr >
			<td >DNA Polymerase I, Klenow</td>
			<td >New England BioLabs</td>
			<td >M0210</td>
			<td >Section 9</td>
		</tr>
		<tr >
			<td >10x NEBuffer 2 (10x Reaction Buffer 2)</td>
			<td >New England BioLabs</td>
			<td >B7002</td>
			<td >Sections 9-11, 13, 15, 20</td>
		</tr>
		<tr >
			<td >Thermomixer C</td>
			<td >Eppendorf</td>
			<td >5382</td>
			<td >Sections 9-16</td>
		</tr>
		<tr >
			<td >ATP</td>
			<td >Roche Life Science</td>
			<td >010419979001</td>
			<td >Sections 9, 11, 13</td>
		</tr>
		<tr >
			<td >dNTPs</td>
			<td >New England BioLabs</td>
			<td >N0447</td>
			<td >Sections 9, 12, 19, 23</td>
		</tr>
		<tr >
			<td >T4 Polynucleotide Kinase</td>
			<td >New England BioLabs</td>
			<td >M0201</td>
			<td >Sections 9, 13</td>
		</tr>
		<tr >
			<td >dATP</td>
			<td >New England BioLabs</td>
			<td >N0440</td>
			<td >Sections 10, 20</td>
		</tr>
		<tr >
			<td >Klenow 3&#39;-5&#39; Exo Minus</td>
			<td >New England BioLabs</td>
			<td >M0212</td>
			<td >Sections 10, 20</td>
		</tr>
		<tr >
			<td >T4 DNA ligase&#160;</td>
			<td >New England BioLabs</td>
			<td >M0202</td>
			<td >Sections 11, 21</td>
		</tr>
		<tr >
			<td >&#934;-29 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0269</td>
			<td >Sections 12, 19</td>
		</tr>
		<tr >
			<td >10x &#934;-29 Buffer</td>
			<td >New England BioLabs</td>
			<td >B0269</td>
			<td >Sections 12, 19</td>
		</tr>
		<tr >
			<td >Lambda Exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0262</td>
			<td >Section 14</td>
		</tr>
		<tr >
			<td >10x Lambda Buffer</td>
			<td >New England BioLabs</td>
			<td >B0262</td>
			<td >Section 14</td>
		</tr>
		<tr >
			<td >RecJf Exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0264</td>
			<td >Section 15</td>
		</tr>
		<tr >
			<td >Proteinase K</td>
			<td >Roche Life Science</td>
			<td >03115828001</td>
			<td >Section 16</td>
		</tr>
		<tr >
			<td >Glycogen</td>
			<td >Roche Life Science</td>
			<td >010901393001</td>
			<td >Section 18</td>
		</tr>
		<tr >
			<td >10x T4 Ligase Buffer</td>
			<td >New England BioLabs</td>
			<td >B0202</td>
			<td >Section 21</td>
		</tr>
		<tr >
			<td >AMPure XP (clean up) beads&#160;</td>
			<td >Beckman Coulter</td>
			<td >A63881</td>
			<td >Section 22</td>
		</tr>
		<tr >
			<td >Q5 Hot Start DNA Polymerase&#160;</td>
			<td >New England BioLabs</td>
			<td >M0493</td>
			<td >Section 23</td>
		</tr>
		<tr >
			<td >5x Q5 Buffer (5x PCR Buffer)</td>
			<td >New England BioLabs</td>
			<td >B9027</td>
			<td >Section 23</td>
		</tr>
		<tr >
			<td >Qubit Fluorometer</td>
			<td >Invitrogen</td>
			<td >Q33216</td>
			<td >Section 25</td>
		</tr>
		<tr >
			<td >QIAquick Gel Extraction Kit</td>
			<td >Qiagen</td>
			<td >28704</td>
			<td >Section 25</td>
		</tr>
		<tr >
			<td >Optical Clear Qubit tubes&#160;</td>
			<td >Invitrogen</td>
			<td >Q32856</td>
			<td >Section 26</td>
		</tr>
		<tr >
			<td >Qubit dsDNA High Sensitivity Assay kit</td>
			<td >Invitrogen</td>
			<td >Q32851</td>
			<td >Section 26</td>
		</tr>
	</tbody>
</table>

the chip-exo method: identifying protein-dna interactions with near base pair precision

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5&#39; DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

The following figures illustrate representative results from the ChIP-exo protocol presented here. In contrast to traditional ChIP-seq methodologies with few enzymatic steps, ChIP-exo requires eleven sequentially dependent enzymatic reactions (Figure 1). Thus, care must be taken at each step to ensure that each reaction component is added to its respective reaction master mix. We recommend generating a formulaic spreadsheet based on the reaction Tables to automatically pe...

Watch this Scientific Journal Video about The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision at JoVE.com

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA ...

The overall goal of this functional genomic technology is to identify the precise genomic locations that a given protein occupies in living cells. This method can help answer key questions in the fields of gene regulation and epigenetics such as how the transcription machinery assembles and operates in the context of chromatin. The main advantage of this technique is it reveals the exact genomic location for protein DNA interactions at near-base pre-resolution with exceptionally low background.Generally, individuals new to this method will struggle because the protocol is complex and requires attention to detail throughout. To sonicate nuclear lysates, begin by placing resin adapters in 15 milliliter polystyrene tubes containing nuclear extracts. Sonicate the lysates in an ice cold water bath at medium power for 30 seconds on and 30 seconds off for a total of two 15-minute sessions.To check sonication results, transfer two 10 microliter samples from each tube into 1.5 milliliter tubes. Then reverse crosslinks by combining the first 10 microliter sample with 10 microliters of TE-RNase A and 0.2 microliters of proteinase K.Incubate the tube at 37 degrees Celsius for 30 minutes. Then add four microliters of 6X xylene DNA dye.To the second 10 microliter sample, preserve crosslinks by adding two microliters of 6X xylene DNA dye. Run both samples on a 1.5%agarose gel at 140 volts for approximately 30 to 45 minutes until the bromophenol dye in the latter is three-quarters of the way down the gel. If sonication was successful, transfer sonicated lysates to two milliliter tubes containing 150 microliters of 10%Triton X-100 and vortex to mix.Pellet the insoluble chromatin and debris by spinning the sonicated lysates at 20, 000 times g at four degrees Celsius for 10 minutes. Then transfer the supernatants to new two milliliter tubes. Store the samples at negative 80 degrees Celsius indefinitely.For each chromatin immunoprecipitation or ChIP reaction, combine 1.5 milliliters of sonicated extracts with antibody bead conjugates in 1.5 milliliter tubes. Incubate the tubes on a mini tube rotator at a speed of nine at four degrees Celsius overnight. Check after a few minutes to ensure samples are not leaking and are mixing properly.The following day, very briefly spin down the tubes to collect the liquid in the caps and place the tubes on a magnetic rack for one minute. Then aspirate the extract. Add 0.75 milliliters of RIPA buffer to each tube.Then remove the tubes from the magnetic rack and invert them several times to mix. After a brief spin, place the tubes back on the magnet and aspirate the supernatant. Following the last wash, add 0.75 milliliters of 10 millimolar Tris HCL to each tube.Remove the tubes from the magnetic rack and invert the samples several times to mix. After a brief spin, place the tubes back on the rack and aspirate the supernatant. After carrying out the polishing and detailing reactions according to the text protocol, fill out the ligation master mix calculations.Then make the ligation mix in a 1.5 milliliter tube on ice and pipette to mix the solution. With the sample tubes on the magnetic rack, add 48 microliters of P7 ligation master mix and two microliters of a different adapter index to each of the sample resins. Incubate the samples in a ThermoMixer at three times g in 25 degrees Celsius for two hours.After washing the samples and performing the Phy 29 nick repair and kinase reactions according to the text protocol, fill out the lambda exonuclease master mix calculations and make the lambda exonuclease mix in a 1.5 milliliter tube on ice. Add 50 microliters of lambda exonuclease mix to each sample resin while still on the magnetic rack. Then incubate the samples in a ThermoMixer at three times g in 37 degrees Celsius for 30 minutes.After aspirating the last wash as described in the text protocol, carry out RecJf nuclease reaction by filling out the RecJf master mix calculations and making the RecJf mix on ice. Add 50 microliters of the RecJf mix to each sample resin on the magnetic rack. Then incubate the samples in a ThermoMixer at three times g in 37 degrees Celsius for 30 minutes.After aspirating the last wash, prepare the ChIP elution buffer and proteinase K master mix and add 200 microliters of the solution to each sample resin. Incubate the samples in a ThermoMixer at three times g in 65 degrees Celsius overnight with a heated lid to prevent condensation. The following day, briefly spin the samples and place the tubes on the magnetic rack for one minute.Then transfer 200 microliters of supernatant to a new 1.5 milliliter tube containing 200 microliters of TE buffer. Following DNA extraction, P7 primary extension and ATL reactions, fill out the P5 adapter ligation table master mix calculations and make the ligation master mix on ice. Add 20 microliters of the ligation mix to each sample and pipette to mix.Then incubate the samples in a thermal cycler at 25 degrees Celsius for two hours. Carry out DNA purification and quantification according to the text protocol. As shown here, it is worth spending time to optimize sonication conditions to obtain high-quality chromatin extracts that yield DNA fragments between 100 to 500 base pairs.And electrophoretic mobility shift assay is used to verify that the sonicated extracts contain intact protein DNA crosslinks, evident as a super shift of DNA fragments. To assess background signal, a mock ChIP is routinely performed without antibody in the reaction. A high-quality ChIP exo-library will yield very little background in the mock sample compared to a ChIP-seq library preparation.Shown here is a bioanalyzer assessment of a ChIP-exo library. Analysis accurately ensures the library size distribution and detects contaminating adapter dimers that run at 125 base pairs. This panel illustrates the smooth distribution of strand-separated ChIP-exo tag five prime Ns for polymerase II, TFIIB and TBP at the human RPS12 gene in proliferating K562 cells.Shown in this figure are average ChIP-exo patterns relative to the transcriptional start sites of protein coding genes. The major peak locations for TBP, TFIIB and polymerase II in the composite plot reflect the genomic organization of human transcription initiation complexes. Once mastered, this technique can be done in four days if performed properly.After its development, this technique paved the way for researchers in the field of gene regulation to explore molecular details of subnuclear structure in assymetry in yeast. Following this procedure, other methods like RNase seek expression profiling can be performed to address questions such as whether a given factor influences the expression of a nearby gene. While attempting this procedure, it's important to remember to carefully fill out master mix tables for all enzymatic steps.Don't forget, working with carcinogens and other caustic agents can be extremely hazardous and precautions such as wearing personal protective equipment should always be taken while performing this procedure. After this watching video, you should have a good understanding of how to prepare a high-quality ChIP-exo library.

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Exploring Cognitive Functions in Babies, Children &amp; Adults with Near Infrared Spectroscopy

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as language1,2,3,4,5,6,7,8,9,10, memory11, and attention12 is underway worldwide involving adults, children and infants 3,4,13,14,15,16,17,18,19 with typical and atypical cognition20,21,22. The contemporary challenge of using fNIRS for cognitive neuroscience is to achieve systematic analyses of data such that they are universally interpretable23,24,25,26, and thus may advance important scientific questions about the functional organization and neural systems underlying human higher cognition.
Existing neuroimaging technologies have either less robust temporal or spatial resolution. Event Related Potentials and Magneto Encephalography (ERP and MEG) have excellent temporal resolution, whereas Positron Emission Tomography and functional Magnetic Resonance Imaging (PET and fMRI) have better spatial resolution. Using non-ionizing wavelengths of light in the near-infrared range (700-1000 nm), where oxy-hemoglobin is preferentially absorbed by 680 nm and deoxy-hemoglobin is preferentially absorbed by 830 nm (e.g., indeed, the very wavelengths hardwired into the fNIRS Hitachi ETG-400 system illustrated here), fNIRS is well suited for studies of higher cognition because it has both good temporal resolution (~5s) without the use of radiation and good spatial resolution (~4 cm depth), and does not require participants to be in an enclosed structure27,28. Participants cortical activity can be assessed while comfortably seated in an ordinary chair (adults, children) or even seated in mom s lap (infants). Notably, NIRS is uniquely portable (the size of a desktop computer), virtually silent, and can tolerate a participants subtle movement. This is particularly outstanding for the neural study of human language, which necessarily has as one of its key components the movement of the mouth in speech production or the hands in sign language. 
The way in which the hemodynamic response is localized is by an array of laser emitters and detectors. Emitters emit a known intensity of non-ionizing light while detectors detect the amount reflected back from the cortical surface. The closer together the optodes, the greater the spatial resolution, whereas the further apart the optodes, the greater depth of penetration. For the fNIRS Hitachi ETG-4000 system optimal penetration / resolution the optode array is set to 2cm. 
Our goal is to demonstrate our method of acquiring and analyzing fNIRS data to help standardize the field and enable different fNIRS labs worldwide to have a common background.

Here we describe a data collection and data analysis method for functional Near Infrared Spectroscopy (fNIRS), a novel non-invasive brain imaging system used in cognitive neuroscience, particularly in studying child brain development. This method provides a universal standard of data acquisition and analysis vital to data interpretation and scientific discovery.

An explosion of functional Near Infrared Spectroscopy (fNIRS) studies investigating cortical activation in relation to higher cognitive processes, such as ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Associated Chromosome Trap for Identifying Long-range DNA Interactions

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure. Each chromosome occupies a specific territory, that may change according to stage of development or cell cycle. Gene expression can occur in specialized transcriptional factories where chromatin segments may loop out from various chromosome territories, leading to co-localization of DNA segments which may exist on different chromosomes or far apart on the same chromosome. The Associated Chromosome Trap (ACT) assay provides an effective methodology to identify these long-range DNA associations in an unbiased fashion by extending and modifying the chromosome conformation capture technique. The ACT assay makes it possible for us to investigate mechanisms of transcriptional regulation in trans, and can help explain the relationship of nuclear architecture to gene expression in normal physiology and during disease states.

The associated chromosome trap (ACT) assay is a novel unbiased method for identifying long-range DNA interactions. The characterization of long range DNA interactions will allow us to determine the relationship of nuclear architecture to gene expression in both normal physiology and in diseased states.

Genetic information encoded by DNA is organized in a complex and highly regulated chromatin structure.  Each chromosome occupies a specific territory, ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...