Name: Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation (Video) | JoVE Journal
Uploaded: 2012-04-30T13:00:00.000+00:00
Duration: 21 min 55 s
Description: Learn ChIA-PET for mapping chromatin interactions and understanding transcription regulation using next-generation sequencing. This method enables genome-wide analysis of long-range chromatin interactions.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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.

<ol><li>Cai, S., Lee, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SATB1+packages+densely+looped%2C+transcriptionally+active+chromatin+for+coordinated+expression+of+cytokine+genes%5BTitle%5D)+AND+%22Nat.+Genet%22%5BJournal%5D)">SATB1 packages densely looped, transcriptionally active chromatin for coordinated expression of cytokine genes.</a> Nat. Genet. 38, 1278-1288 (2006).</li>
<li>Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+organization+of+replication+and+transcription%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The organization of replication and transcription.</a> Science. 284, 1790-1795 (1999).</li>
<li>Das, P. M., Ramachandran, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+immunoprecipitation+assay%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Chromatin immunoprecipitation assay.</a> Biotechniques. 37, 961-969 (2004).</li>
<li>Deng, W., Blobel, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do+chromatin+loops+provide+epigenetic+gene+expression+states%5BTitle%5D)+AND+%22Curr.+Opin.+Genet.+Dev%22%5BJournal%5D)">Do chromatin loops provide epigenetic gene expression states.</a> Curr. Opin. Genet. Dev. 20, 548-554 (2010).</li>
<li>Fullwood, M. J., Han, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+interaction+analysis+using+paired-end+tag+sequencing%5BTitle%5D)+AND+%22Curr.+Protoc.+Mol.+Biol%22%5BJournal%5D)">Chromatin interaction analysis using paired-end tag sequencing.</a> Curr. Protoc. Mol. Biol. Chapter 21, 21-25 (2010).</li>
<li>Fullwood, M. J., Liu, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+oestrogen-receptor-alpha-bound+human+chromatin+interactome%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">An oestrogen-receptor-alpha-bound human chromatin interactome.</a> Nature. 462, 58-64 (2009).</li>
<li>Jackson, D. A., Hassan, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+focal+sites+of+transcription+within+human+nuclei%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Visualization of focal sites of transcription within human nuclei.</a> EMBO J. 12, 1059-1065 (1993).</li>
<li>Li, G., Fullwood, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ChIA-PET+tool+for+comprehensive+chromatin+interaction+analysis+with+paired-end+tag+sequencing%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">ChIA-PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing.</a> Genome Biol. 11, R22(2010).</li>
<li>Li, G., Ruan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extensive+Promoter-Centered+Chromatin+Interactions+Provide+a+Topological+Basis+for+Transcription+Regulation%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation.</a> Cell. 148, 84-98 (2012).</li>
<li>Ng, P., Wei, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paired-end+diTagging+for+transcriptome+and+genome+analysis%5BTitle%5D)+AND+%22Curr.+Protoc.+Mol.+Biol.+Chapter%22%5BJournal%5D)">Paired-end diTagging for transcriptome and genome analysis.</a> Curr. Protoc. Mol. Biol. Chapter. Chapter 21, 12(2007).</li>
<li>Nowak, D. E., Tian, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two-step+cross-linking+method+for+identification+of+NF-kappaB+gene+network+by+chromatin+immunoprecipitation%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.</a> Biotechniques. 39, 715-725 (2005).</li>
<li>Osborne, C. S., Chakalova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+genes+dynamically+colocalize+to+shared+sites+of+ongoing+transcription%5BTitle%5D)+AND+%22Nat.+Genet%22%5BJournal%5D)">Active genes dynamically colocalize to shared sites of ongoing transcription.</a> Nat. Genet. 36, 1065-1071 (2004).</li>
<li>Rippe, K., von Hippel, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Action+at+a+distance%3A+DNA-looping+and+initiation+of+transcription%5BTitle%5D)+AND+%22Trends+Biochem.+Sci%22%5BJournal%5D)">Action at a distance: DNA-looping and initiation of transcription.</a> Trends Biochem. Sci. 20, 500-506 (1995).</li>
<li>Schoenfelder, S., Sexton, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preferential+associations+between+co-regulated+genes+reveal+a+transcriptional+interactome+in+erythroid+cells%5BTitle%5D)+AND+%22Nat.+Genet%22%5BJournal%5D)">Preferential associations between co-regulated genes reveal a transcriptional interactome in erythroid cells.</a> Nat. Genet. 42, 53-61 (2010).</li>
<li>Zeng, P. Y., Vakoc, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+dual+cross-linking+for+identification+of+indirect+DNA-associated+proteins+by+chromatin+immunoprecipitation%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation.</a> Biotechniques. 41, 694-698 (2006).</li>
</ol>

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.

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

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.

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.

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

COMIN Interaction analysis using powder and tag sequencing is a method for genome wide comin long range comin interaction analysis. In short, we call this magic share pad. This method involves many molecular biology steps and can be complicated.Therefore, in this following videotape, we'll show you some of the key steps involved in com, immuno immunoprecipitation, and the library construction for the chair. PET analysis Chromatin immunoprecipitation known as chip, is the first critical step in constructing a chair pet library. Here, chromatin samples from cell cultures are cross-linked sonicated and immuno precipitated.Separate aliquots of chip DNA are then ligated to barcoded half linkers and proximity ligation is carried out. Pets are released by restriction digestion, purified on coated magnetic beads, and li gated to adapters for next generation sequencing. Chimeric chair pets are identified via the AB linker composition and tag flanking.The central linker sequence are read out and mapped to the genome. In this video, we will be showing the standard chip protocol routinely used in our lab on a brass cancer cell line MCF seven From our data, higher chip enrichment with specific antibodies yields better chair pet data. Hence, we strongly recommend optimization of the chip protocol and enrichment validation by QPCR.Before proceeding to chair pet library Construction CF seven cells are cultured to about 75%co fluency in a 500 centimeter square tissue culture dish, which contains approximately 20 million cells before cross-linking. Discard growth media and wash cells twice. With warm PBS fix Cells with freshly prepared 1.5 millimolar EGS for 45 minutes on a rotator at room Temperature At 37%formaldehyde to a final concentration of 1%and incubate for 20 minutes.Crosslinking efficiency is critical and varies with cell lines and the protein of interest. Crosslink a concentration incubation time and temperature should be optimized. Quench the cross linkers with a final concentration of 200 millimolar glycine for 10 Minutes.Discard quenched cross linkers and washed twice with cold PBS Hover cells by scraping pallet cells with a refrigerated benchtop centrifuge, The pallet is now ready for cell lysis. Alternatively, freeze palate at minus 80 degrees Celsius. If cell palate Is frozen, thaw on ice before proceeding with cell lysis resand palate with 0.1%SDS lysis Buffer.Rotate the tube at four degrees Celsius palate with a refrigerated centrifuge, discard Supernatant and repeat cell lysis. Once nuclear lysis is performed to release cross-linked chromatin before chromatin fragmentation. The absence of nuclear membrane allows the cross-linked chromatin to be sonicated under gentler conditions.Reus Band nuclear palette in 1%SDS lysis Buffer. Transfer the suspension to a high speed centrifuge tube. Rotate the tube at four degrees Celsius.Remove a nuclear debris via centrifugation. The chromatic palette should look like this. Use a P 1000 piper tip to break up the palette into pieces and wash twice with 0.1%SDS lysis Buffer.Rotate the tube at four degrees Celsius palate with a refrigerated centrifuge. The nuclear palate Is now ready for sonication. Alternatively, freeze palate at minus 80 degrees Celsius.Transfer palate to a clear tube Add 0.1%SDS lysis, buffer to the palate and remove all bubbles. Keep samples cold throughout sonication to prevent overheating. Different conditions such as buffer volume, probe depth, sonication strength, number of cycles and extent of cross-linking can influence sonication efficiency.Reverse cross-link and aliquot of chromatin using proteinase K and run an aros gel. To verify fragmentation efficiency optimization is recommended to obtain the desired DNA size range between 200 to 600. Base pair remove cell dri by centrifugation.The chromatin is now ready to be pre-cleared with magnetic beads. Alternatively stopped at minus 80 degrees Celsius until sufficient chromatin is collected. To start a chip Wash beads three times with beads wash buffer this and future washes involving magnetic protein beads consist of the following steps.Add sonicated chromatin to the pre-washed beads and incubate overnight at four degrees Celsius in a separate tube. Coat the pre-washed beads with antibody and incubate overnight at four degrees Celsius. Wash the antibody coated beads twice with beads wash buffer.The high Complexity of substrate for proximity ligation inevitably leads to substantial non-specific noise, thereby increasing the cost of sequencing. Apart from reducing the level of complexity and background noise. We used a mouse RNA polymerase two monoclonal antibody to identify chromatin interactions involved in transcription regulation.This allows the investigation of active promoters and their corresponding regulatory regions, allowing the understanding of efficient and possibly cooperative transcription. Discard wash buffer from the antibody Coated beads combined Pre-cleared chromatin with antibody coated beads and incubate overnight at four degrees Celsius after overnight incubation, the beads are subjected to a series of washes to remove unbound chromatin resand beads In TE buffer aliquot. A small amount for quantitative And enrichment check chromatin interaction analysis using pad and tag sequencing is a technique developed for large scale de novo analysis of higher order chromatin structures interacting.DNA fragments which have been enriched by chromatin immunoprecipitation are captured by proximity ligation. The pad and tag strategy is applied to the construction of CHI PET Libraries, which are sequenced by high throughput next generation sequencing technologies. In this video, we will show the critical experimental aspects in CHI PET library construction.The first half of the library construction involves manipulating protein A or G beats, which the DNA fragments are bound to, to remove the enzymes and buffering salt. After each enzymatic reaction, use gentle centrifugation and flicking action for each washing step. Pipette the supernatant several times to maximize the capture of beads when placed on a magnetic particle.Concentrator for all incubation steps containing beads in the reaction mixture. Prepare master mix beforehand and add enzyme last after the beads are resus resuspended homogeneously Ensure that the beads are well mixed throughout the entire incubation. Half linker oligo nucleotides are designed with an internal barcode consisting of four nucleotides and recognition site for the type two S restriction enzyme MME one.After ligating the half linkers to the tethered DNA fragments in C chromatic complexes, the interacting DNA fragments will be connected by a complete linker sequence. These half linkers generate sequences with heterodimer ab linkers derived from non-specific ligation products or sequences with homodimer, AA or BB linkers derived from specific ligation products. Hence the use of two half linkers with different nucleotide barcodes allow specifications of different experiments or replicates as well as monitoring of the non-specific chimeric ligation rates between different chip complexes.It is important to set up the reaction on ice And thaw the half linkers gently on ice. Combine half linkers well with water and then with a PEG containing ligase buffer. Ensure the master mix is well mixed before resus.Suspending the chip DNA add T four DNA ligase only after the half linkers and chip DNA fragments a well mixed to prevent concatenation of half linkers Seal the tube with parfum Incubate overnight at 16 degrees Celsius with rotation. Intact Chromatin complexes are then alluded off the bead through the addition of SDS at buffer EB to eluted DNA to lower the SDS concentration at Triton X 100 to quench all the SDS circularization of chromatin complexes is facilitated by the ligation of two half linkers, creating a complete linker sequence at the interaction junction. Ligate elute chromatin complexes at extremely dilute conditions to minimize ligations between different DNA protein complexes at T four DNA ligase only after the eluted chromatic complexes have been well diluted in the reaction mixture After Reverse cross-linking To remove DNA associated protein purifies circularized DNA fragments using phenyl chloroform Extraction, Use KaiGen phase lock gel system for easy separation.After phenyl extraction, Use glyco blue A DNA carrier to increase mass and visibility of the palate. Half linker oligo nucleotides contain flanking MM E one recognition sites. MM E one A Type two S restriction enzyme cuts 18 and 20 base pairs downstream of its target binding sites to generate short tags of the chromatin fragment producing PET tag linker tag constructs abbreviated as pets.In addition, each half linker is modified with biotin to enable purification of pet constructs by streptavidin coated magnetic bead. Perform MME one digestion to release tag Linker constructs. Purify the pets by selective binding to strapped Aden beads ligate the pet constructs with adapters for high throughput sequencing.Use fusion DNA polymerase to amplify the CHI PET Library Fusion. DNA polymerase is used to generate templates with high accuracy and efficiency. Set up PCR reaction cold and add fusion DNA polymerase last.As the enzyme exhibits three prime to five prime exonuclease activity that can degrade primers in the absence of DN Tps. If the chair pet library is successfully constructed, there should be a prominent, well-defined band of approximately 223 base pairs. Avoid non-specific bands when excising the 223 base pair band from the gel and purify the excised band using the Gel Crush Protocol.Perform a Quality check using Agilent 2, 100 bioanalyzer with a DNA 1000 kit before high throughput sequencing. We have just demonstrated the CHI PET procedure for unbiased detection of genome-wide chromatin interactions. The quality of CHI PET data depends on the effectiveness of the chip antibody used and sequencing depth.Here we use 18 to 20 million reads for interaction mapping. This specific protocol has been used to successfully characterize chromatin interaction networks bound by the estrogen receptor and RNA polymerase in the human genome. We foresee this assay to be a powerful tool for future studies after the three dimensional architecture of transcription biology in whole genome context.We hope this video will be useful to you. Thank you for watching.I.

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

Research

JoVE Journal

Biology

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.

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

The question of how chromatin regulators and chromatin states affect the genome in vivo is key to our understanding of how early cell fate decisions are made in the developing embryo. ChIP-Seq—the most popular approach to investigate chromatin features at a global level—is outlined here for Xenopus embryos.

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

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.

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

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

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

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

Here, we describe methods to perform ChIP-Seq and CARIP-Seq, including library preparation for next-generation sequencing, to generate global epigenomic and chromatin-associated RNA maps in ES cells.

CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

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

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

Here, we present a quick and simple ATAC-seq protocol to be implemented in L4-stage Caenorhabditis elegans whole-worm using only 30 µL of worm pellet.

Chromatin Accessibility Profiling in Whole Caenorhabditis elegans L4 Larvae

Here, we present a protocol to capture RNA-associated chromatin DNA-DNA interactions for mapping RNA-related three-dimensional genome organization.

RNA-Associated Chromatin DNA-DNA Interaction Method

Learn ChIA-PET for mapping chromatin interactions and understanding transcription regulation using next-generation sequencing. This method enables genome-wide analysis of long-range chromatin interactions.

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

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

Explore More Videos