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 Views.  Agency for Science, Technology and Research, Singapore. 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.

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

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

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 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).