The authors are supported by A*STAR of Singapore. In addition, M.J.F. is supported by an A*STAR National Science Scholarship, a L'Oreal For Women in Science National Fellowship and a Lee Kuan Yew Post-Doctoral Fellowship. Y.R. is supported by NIH ENCODE grants (R01 HG004456-01 and R01 HG003521-01). The authors also acknowledge the videography team of 8 Pixels Productions, Singapore, in particular Mr. Kelvin Issey, Mr. Chang Kai Xiang and Mr. Sherwin Gan for filming the scenes, Ms. Siti Rahim for video editing and Ms. Michelle Teo for the voice-over.

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No conflicts of interest declared.

ChIA-PET is a method developed to identify long-range interactions in transcription regulation. One of the critical factors that determine the quality of a ChIA-PET library is the quality of ChIP material.
The protocol shown in the video incorporates the use of EGS and formaldehyde to cross-link the cells. The use of formaldehyde combined with a second cross-linking reagent bearing a longer spacer arm may help in the binding of proteins which could not be bound by formaldehyde alone 3,11,15. We have constructed libraries with this method which have demonstrated robust binding sites and long-range interactions 9. However, the cross-linking and ChIP conditions should be optimized for each factor of interest, and it is important not to over-crosslink as too much cross-linking will result in difficulties in fragmentation by sonication, and may possibly result in spurious chromatin interactions. Chromatin interactions identified by ChIA-PET should be validated by a different method, such as fluorescence in-situ hybridization 4. 
We recommend a minimum of 100 ng of chromatin material. While we have constructed good quality libraries from 50 ng of chromatin material, we have observed that large amounts of starting material allowed construction of ChIA-PET libraries with less than 16 PCR cycles, thereby minimizing amplicons and redundancy of each library. This lower redundancy correlated with higher unique mapped tags and also a high percentage of usable data, thereby enabling a more comprehensive chromatin interaction map with fewer lanes of sequencing. The final packed volume of beads in each tube should be 50 &mu;l and 100 &mu;l for magnetic and Sepharose beads respectively. If the packed bead volume is less than stated, bring to the minimum packed volume with similarly pre-cleared blank magnetic or Sepharose beads to minimize loss of DNA-bearing beads in subsequent steps. Sawed-off tips or large-core tips should be used for pipetting Sepharose beads. 
The following modifications were incorporated following the previously published ChIA-PET protocol 5. Firstly, magnetic G beads were used to minimize sample loss during washes. In addition, we identified non-specific bands with approximate sizes of 100 bp and 138 bp to be amplicons of self-ligated half-linkers or/and adaptors. Hence, we reduced the concentration of biotinylated half-linkers and 454 GS20 adaptors to minimize non-specific bands during PCR amplification. The proximity ligation volume was reduced from 50 ml to 10 ml to minimize sample loss during subsequent purification steps and also save on reagent costs. We also increased the incubation time to immobilize ChIA-PET DNA to beads to ensure maximal capture of ChIA-PET DNA on the streptavidin beads.
During the proximity ligation step, chimeric ligations that do not represent true in vivo chromatin interactions are inevitably generated in a non-specific and random manner. Hence, to evaluate the quality of data from any ChIA-PET experiment, the rate of chimerism is estimated from the use of two different half-linkers with specific nucleotide barcodes TAAG and ATGT 5. After high-throughput sequencing, the ChIA-PET sequences are first analyzed for linker barcode composition and sequences derived from specific ligation products and non-specific ligation products can be distinguished 8. The percentage of known chimeras (i.e. heterodimers AB linkers) present in our in-house MCF-7 RNA Polymerase II ChIA-PET libraries is less than 15%.
ChIA-PET sequences are subsequently classified into two categories, namely self-ligation PETs and inter-ligation PETs. Self-ligation PETs are obtained from self-circularization ligation of the chromatin fragments while inter-ligation PETs are derived from inter-ligation between two different DNA fragments. The latter is then sub-divided into three different categories based on the genomic distance of each tag on the same chromosome (intrachromosomal inter-ligation PETs) or that both tags are mapped to two different chromosomes (interchromosomal inter-ligation PETs). We have developed a ChIA-PET tool software package to sort out the different categories 8. This will be based on the DNA fragments that are in the library. Generally, smaller ChIP fragments will give a higher resolution and the cut off for these RNA Polymerase II ChIA-PET libraries is about 4 kb.
In addition, real chromatin interactions can be distinguished from random noise by counting the number of inter-ligation PETs in an interaction cluster; in other words, a cluster of high PET count is said to have a higher probability of being a real chromatin interaction 8. 
 To filter our false positives arise from highly enriched anchors which can form inter-ligation PETs by random chance, a statistical analysis framework has also been formulated to account for random formation of any inter-ligation PETs between two anchors 8.
In conclusion, the ChIA-PET technique allows mapping chromatin interaction networks on a global scale. The implementation of ChIP in ChIA-PET allows the reduction of library complexity and background noise. In addition, ChIP adds specificity to chromatin interactions, enabling the examination of specific chromatin interactions associated with particular transcription factors 5.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>4-20% gradient TBE gel</td>
			<td>Invitrogen</td>
			<td>EC6225BOX</td>
			<td>Step B, 6.3 </td>
		</tr>
		<tr>
			<td>5 x T4 DNA Ligase Buffer with PEG</td>
			<td>Invitrogen</td>
			<td>46300018</td>
			<td>Step B, 2.2</td>
		</tr>
		<tr>
			<td>6% TBE gel</td>
			<td>Invitrogen</td>
			<td>EC6263BOX</td>
			<td>Step B, 6.8</td>
		</tr>
		<tr>
			<td>Agilent DNA 100 Assay </td>
			<td>Agilent Technologies </td>
			<td>5067-1504</td>
			<td rowspan="3">Step B, 7.1</td>
		</tr>
		<tr>
			<td>Agilent High Sensitivity DNA Assay</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
		</tr>
		<tr>
			<td>Agilent Bioanalyzer 2100</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
		</tr>
		<tr>
			<td>Centrifuge Tube Filters (Spin-X)</td>
			<td>Corning</td>
			<td>CLS8160</td>
			<td>Step B, 3.7 and 6.9</td>
		</tr>
		<tr>
			<td>Dark Reader Transilluminator</td>
			<td>Clare Chemical Research</td>
			<td>DR46B</td>
			<td>Step B, 6.8</td>
		</tr>
		<tr>
			<td>Digital Sonifier Cell Disrupter</td>
			<td>Branson</td>
			<td>450D-0101063591</td>
			<td>Step A, 4.3</td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet (Magnetic Particle Concentrator)</td>
			<td>Invitrogen</td>
			<td>123-21D</td>
			<td>Step A, 7.5.1 Step B, for all magnetic beads washing steps</td>
		</tr>
		<tr>
			<td>DynaMag-15 Magnet (Magnetic Particle Concentrator)</td>
			<td>Invitrogen</td>
			<td>123.01D</td>
			<td>Step A, 5 and 6</td>
		</tr>
		<tr>
			<td>DynaMag-PCR (Magnetic Particle Concentrator)</td>
			<td>Invitrogen</td>
			<td>49-2025</td>
			<td>Step B, 6.5</td>
		</tr>
		<tr>
			<td>Escherichia coli DNA Polymerase I</td>
			<td>NEB</td>
			<td>M0209</td>
			<td>Step B, 5.6</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9516</td>
			<td>Step A, 7.5.1 Step B, 4.3 and 6.5</td>
		</tr>
		<tr>
			<td>Illumina cBot Cluster Generation System</td>
			<td>Illumina</td>
			<td>SY-301-2002</td>
			<td>Step B, 7.4</td>
		</tr>
		<tr>
			<td>Illumina Genome Analyzer IIx</td>
			<td>Illumina</td>
			<td>SY-301-1301</td>
			<td>Step B, 7.5</td>
		</tr>
		<tr>
			<td>Illumina PE primers</td>
			<td>Illumina</td>
			<td>PE-102-1004</td>
			<td>Step B, 5.4 (if necessary)</td>
		</tr>
		<tr>
			<td>Intelli-Mixer </td>
			<td>Palico Biotech</td>
			<td>RM-2L</td>
			<td>Step B, any incubations with rotation</td>
		</tr>
		<tr>
			<td>LightCycler 480 Real-Time PCR System</td>
			<td>Roche </td>
			<td>04 640 268 001</td>
			<td>Step A, 7.5.3 Step B, 7.3</td>
		</tr>
		<tr>
			<td>LightCycler480 DNA SYBR Green I MasterMix</td>
			<td>Roche</td>
			<td>03 752 186 001</td>
			<td>Step B, 7.5.3</td>
		</tr>
		<tr>
			<td>M-280 Streptavidin Dynabeads</td>
			<td>Invitrogen</td>
			<td>11206D</td>
			<td>Step B, 5.2</td>
		</tr>
		<tr>
			<td>Magnetic (Dynabeads) Protein G</td>
			<td>Invitrogen</td>
			<td>100.03D</td>
			<td>Step A, 5.1.1 and 5.2.1</td>
		</tr>
		<tr>
			<td>MaXtract High Density (2ml)</td>
			<td>Qiagen</td>
			<td>129056</td>
			<td>Step A, 7.5.1</td>
		</tr>
		<tr>
			<td>MaXtract High Density (50ml)</td>
			<td>Qiagen</td>
			<td>129073</td>
			<td>Step B, 4.2</td>
		</tr>
		<tr>
			<td>MmeI</td>
			<td>NEB</td>
			<td>R0637</td>
			<td>R0637</td>
		</tr>
		<tr>
		 <td>RNase ONE Ribonuclease</td>
		 <td>Promega</td>
		 <td>M426C</td>
		 <td>Step B, 4.8</td>
	 </tr>
		<tr>
			<td>RNA Polymerase II (8WG16) monoclonal antibody</td>
			<td>Covance</td>
			<td>MMS-126R </td>
			<td>Step A, 5.2.4</td>
		</tr>
		<tr>
			<td>Oak Ridge Centrifuge Tubes (Polypropylene)</td>
			<td>Nalgene</td>
			<td>3119-0050</td>
			<td>Step A, 3.1</td>
		</tr>
		<tr>
			<td>Oak Ridge Centrifuge Tubes (Teflon FEP)</td>
			<td>Nalgene</td>
			<td>3114-0050</td>
			<td>Step B, 4.3</td>
		</tr>
		<tr>
			<td>Phusion High Fidelity Master Mix</td>
			<td>Finnzymes</td>
			<td>F-531</td>
			<td>Step B, 6</td>
		</tr>
		<tr>
			<td>Picogreen (Quant-iT) dsDNA Reagent</td>
			<td>Invitrogen</td>
			<td>P11495</td>
			<td>Step A, 7.5.2</td>
		</tr>
		<tr>
			<td>Polystyrene Round Bottom Test Tube</td>
			<td>BD Biosciences</td>
			<td>352057</td>
			<td>Step A, 4.1</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets (cOmplete, EDTA-free)</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td>Step A, 1.6 onwards</td>
		</tr>
		<tr>
			<td>Proteinase K Solution (20mg/ml)</td>
			<td>Fermentas</td>
			<td>E00491</td>
			<td>Step A, 4.4, 7.5.1 
			Step B, 4.1</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Fermentas</td>
			<td>EL0013</td>
			<td>Step B, 2.2, 3.9 and 5.4</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase Buffer (NEB)</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td>Step B, 3.3 and 3.9</td>
		</tr>
		<tr>
			<td>T4 DNA Polymerase</td>
			<td>Promega</td>
			<td>M4215</td>
			<td>Step B, 1.2</td>
		</tr>
		<tr>
			<td>T4 DNA Polynucleotide Kinase</td>
			<td>NEB</td>
			<td>M0201</td>
			<td>Step B, 3.3</td>
		</tr>
		<tr>
			<td>TruSeq SBS Kit v5&#x2013;GA</td>
			<td>Illumina</td>
			<td>FC-104-5001</td>
			<td>Regents for Illumina Genome Analyzer IIx system</td>
		</tr>
		<tr>
			<td>TruSeq PE Cluster Kit v2&#x2013;cBot&#x2013;GA</td>
			<td>Illumina</td>
			<td>PE-300-2001</td>
			<td>to be use with Illumina cBot Cluster Generation System</td>
		</tr>
		<tr>
			<td>SYBR Green I </td>
			<td>Invitrogen</td>
			<td>S-7585</td>
			<td>Step B, 6.3 and 6.8</td>
		</tr>
		<tr>
			<td>XCell SureLock Mini-Cell Electrophoresis System</td>
			<td>Invitrogen</td>
			<td>EI0001</td>
			<td>Step B, 6.3 and 6.8</td>
		</tr>
	</tbody>
</table>
<table border="1">
	<tbody>
		<tr>
			<td>Name </td>
			<td colspan="2">Sequence</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td rowspan="2">Biotinylated half-linkers A (200ng/&mu;l)</td>
			<td>Top</td>
			<td>5' GG CCG CGA/iBiodT/ ATC TTA TCC AAC 3'</td>
			<td>250 nmole scale HPLC Purified Internal Biotin dT(9)</td>
		</tr>
		<tr>
			<td>Bot</td>
			<td>5' GTT GGA TAA GAT ATC GC 3'</td>
			<td>250 nmole scale HPLC Purified</td>
		</tr>
		<tr>
			<td rowspan="2">Biotinylated half-linkers B (200ng/&mu;l)</td>
			<td>Top</td>
			<td>5' GGC CGC GA/iBiodT/ ATA CAT TCC AAC 3'</td>
			<td>250 nmole scale HPLC Purified Internal Biotin dT(9)</td>
		</tr>
		<tr>
			<td>Bot</td>
			<td>5' GTT GGA ATG TAT ATC GC 3'</td>
			<td>250 nmole scale HPLC Purified</td>
		</tr>
		<tr>
			<td rowspan="2">Non-biotinylated half-linkers (200ng/&mu;l)</td>
			<td>Top</td>
			<td>5' GGC CGC GAT ATC GGA TCC AAC 3'</td>
			<td>250 nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td>Bot</td>
			<td>5' GTT GGA TCC GAT ATC GC 3'</td>
			<td>250 nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td rowspan="2">GS20 Adaptor A (200ng/&mu;l)</td>
			<td>Top</td>
			<td>5' CCA TCT CAT CCC TGC GTG TCC CAT CTG TTC CCT CCC TGT CTC AGN N 3'</td>
			<td>250 nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td>Bot</td>
			<td>5'CTG AGA CAG GGA GGG AAC AGA TGG GAC ACG CAG GGA TGA GAT GG 3'</td>
			<td>250 nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td rowspan="2">GS20 Adaptor B (200ng/&mu;l)</td>
			<td>Top</td>
			<td>5' CTG AGA CAC GCA ACA GGG GAT AGG CAA GGC ACA CAG GGG ATA GG 3'</td>
			<td>250 nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td>Bot</td>
			<td>5' CCT ATC CCC TGT GTG CCT TGC CTA TCC CCT GTT GCG TGT CTC AGN N 3'</td>
			<td>250 nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td rowspan="2">Illumina-NN adaptors</td>
			<td>Top</td>
			<td>5' ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC 3'</td>
			<td>250 nmole scale HPLC purified See Step B, 5.4</td>
		</tr>
		<tr>
			<td>Bot</td>
			<td>5' phos - GAT CGG AAG AGC GGT TCA GCA GGA ATG CCG AG 3'</td>
			<td>250 nmole scale HPLC purified Phosphorylated 5' end See Step B, 5.4</td>
		</tr>
		<tr>
			<td>Illumina 1-454 (forward) primer (10 &mu;M)</td>
			<td colspan="2">5' AAT GAT ACG GCG ACC ACC GAG ATC TAC ACC CTA TCC CCT GTG TGC CTT G 3'</td>
			<td>250 nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td>Illumina 2-454 (reverse) primer (10 &mu;M)</td>
			<td colspan="2">5' CAA GCA GAA GAC GGC ATA CGA GAT CGG TCC ATC TCA TCC CTG CGT GTC 3'</td>
			<td>250 nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td>qPCR Primer 1.1 (10 &mu;M)</td>
			<td colspan="2">5' AAT GAT ACG GCG ACC ACC GAG AT 3'</td>
			<td>10nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td>qPCR Primer 2.1 (10 &mu;M)</td>
			<td colspan="2">5' CAA GCA GAA GAC GGC ATA CGA 3'</td>
			<td>10nmole scale PCR Grade</td>
		</tr>
		<tr>
			<td>Illumina 3-454 sequencing primer (100 &mu;M)</td>
			<td colspan="2">5'TGC GTG TCC CAT CTG TTC CCT CCC TGT CTC AG 3'</td>
			<td>100nmole scale HPLC Purified</td>
		</tr>
		<tr>
			<td>Illumina 4-454 sequencing primer (100 &mu;M)</td>
			<td colspan="2">5'GTG CCT TGC CTA TCC CCT GTT GCG TGT CTC AG 3'</td>
			<td>100nmole scale HPLC Purified</td>
		</tr>
	</tbody>
</table>
Table of oligos and adaptors. Order oligos from Integrated DNA Technologies (IDT) and prepare linkers and adaptors in the same manner as described previously10. Half-linkers and adaptors may be prepared beforehand and stored for several months at -20 &deg;C.

chromatin interaction analysis with paired-end tag sequencing (chia-pet) for mapping chromatin interactions and understanding transcription regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Watch this Scientific Journal Video about Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation at JoVE.c

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

chromatin-interaction-analysis-with-paired-end-tag-sequencing-chia

Research

JoVE Journal

Biology

30.5K ビュー.  Agency for Science, Technology and Research, Singapore. ペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用分析のための方法です。 de novoの検出。

ビデオ: Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation