게시된 데이터와 귀중한 토론을 공유해 주신 Rhee 연구소의 회원에게 감사드립니다. 이 작품은 캐나다 자연 과학 및 공학 연구 위원회 (NSERC) RGPIN-2018-06404 (H.R.)에 의해 지원되었다.

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+structure+and+organization+of+eukaryotic+pre-initiation+complexes.">Genome-wide structure and organization of eukaryotic pre-initiation complexes.</a> Nature. 483 (7389), 295-301 (2012).</li><li>Zhou, X. F., Yan, Q., Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+regulon+of+a+GntR+family+regulator+via+transcriptome+and+ChIP-exo+analyses+and+its+contribution+to+virulence+in+Xanthomonas+citri.">Deciphering the regulon of a GntR family regulator via transcriptome and ChIP-exo analyses and its contribution to virulence in Xanthomonas citri.</a> Molecular Plant Pathology. 18 (2), 249-262 (2017).</li><li>Kim, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+assessment+of+transcriptional+regulation+on+central+carbon+metabolism+by+Cra+and+CRP.">Systems assessment of transcriptional regulation on central carbon metabolism by Cra and CRP.</a> Nucleic Acids Research. 46 (6), 2901-2917 (2018).</li><li>Niu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome-wide+binding+interactions+of+mouse+and+human+constitutive+androstane+receptors+reveal+novel+gene+targets.">In vivo genome-wide binding interactions of mouse and human constitutive androstane receptors reveal novel gene targets.</a> Nucleic Acids Research. 46 (16), 8385-8403 (2018).</li><li>Uuskula-Reimand, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topoisomerase+II+beta+interacts+with+cohesin+and+CTCF+at+topological+domain+borders.">Topoisomerase II beta interacts with cohesin and CTCF at topological domain borders.</a> Genome Biology. 17, (2016).</li><li>Rhee, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Terminal+Effector+Genes+in+Mammalian+Neurons+Is+Maintained+by+a+Dynamic+Relay+of+Transient+Enhancers.">Expression of Terminal Effector Genes in Mammalian Neurons Is Maintained by a Dynamic Relay of Transient Enhancers.</a> Neuron. 92 (6), 1252-1265 (2016).</li><li>Pugh, B. F., Venters, B. 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저자는 공개 할 것이 없습니다.

이 프로토콜에서, 외핵증 소화뒤에 는 초고매핑 해상도에서 포유류 세포에서 단백질-DNA 상호 작용의 식별을 위한 DNA 라이브러리를 얻기 위하여 이용되는 외핵증 소화에 선행된 ChIP. 많은 변수가 ChIP 엑소 실험의 품질에 기여합니다. 중요한 실험 파라미터는 항체의 질, 초음파 처리의 최적화 및 LM-PCR 주기의 수를 포함합니다. 이러한 중요한 실험 파라미터는 ChIP 엑소 실험을 제한할 수 있으며 아래?...

단백질 DNA 상호 작용의 위치는 유전자 조절의 기계장치에 통찰력을 제공합니다. 높은 처리량 시퀀싱(ChIP-seq)과 결합된 크로마틴 면역 침전은 살아있는 세포1,2에서게놈-DNA 상호 작용을 검사하기 위해 10년 동안 사용되어 왔다. 그러나 ChIP-seq 방법은 낮은 매핑 해상도, 거짓 긍정, 부재중 전화 및 비특이적 배경 신호로 이어지는 DNA 단편화 및 불바운드 DNA 오염의 이질성에 의해 제한됩니다. ChIP와 엑소뉴클레아제 소화(ChIP-exo)의 조합은 ChIP-seq 방법을 단백질-DNA 교차 링크 점으로 트리밍하여, 근거리 쌍 분해능 및 낮은 배경 신호3,4,5를제공한다. ChIP 엑소에서 제공하는 매핑 해상도와 낮은 배경은 게놈 전반에 걸쳐 정확하고 포괄적인 단백질 DNA 결합 위치를 결정할 수 있게 합니다. ChIP-exo는 기능적으로 뚜렷한 DNA 결합 모티프, 전사 인자(TF) 및 다중 단백질-DNA 교차 링크 부위를 특정 게놈 결합 위치에서드러낼 수 있으며, 다른 게놈 매핑 방법에 의해 검출할 수 없는3,4,6,7.
ChIP-exo는 처음에 신진 효모에서 TFs의 서열 특이적 DNA 결합을 검사하고, 게놈4,8,9를통해 개별 히스톤의 전사 사전 개시 복합체 및 서브 뉴클레오소말 구조의 정확한 조직을 연구하기 위해 사용되었다. 2011년4년도입 이후, ChIP-exo는 박테리아, 마우스, 인간 세포7,10,11,12,13,14,15,16,17등많은 다른 유기체에서 성공적으로 활용되고있다. 2016년, Rhee 외14는 다른 프로그래밍 TF Isl1을 이용한 이성구복합체를 형성하는 프로그래밍 TF Lhx3의 다운규제 이후 뉴런 유전자 발현이 어떻게 유지되었는지 이해하기 위해 포유류 뉴런에 ChIP-exo를 처음으로 사용했다. 이 연구는 Lhx3의 부재에서, Isl1는 뉴런 이펙터 유전자 유전자의 유전자 발현을 유지하기 위하여 Onecut1 TF에 의해 결합된 새로운 신경 강화에 모집된다는 것을 보여주었습니다. 이 연구에서 ChIP-exo는 여러 TF가 거의 뉴클레오티드 매핑 해상도에서 복합방식으로 세포 유형 및 세포 단계별 DNA 조절 요소를 동적으로 인식하는 방법을 밝혔습니다. 그밖 연구 결과는 또한 그밖 포유류 세포주에서 단백질과 DNA 사이 상호 작용을 이해하기 위하여 ChIP 엑소 방법을 이용했습니다. 한일(7)은 ChIP-exo를 사용하여 ChIP-exo를 사용하여 마우스 에리스로이드 세포에서 GATA1 및 TAL1 TFs의 게놈 전체 조직을 검사하였다. 이 연구는 TAL1이 적혈구 분화를 통해 GATA1과의 단백질 단백질 상호 작용을 간접적으로 통해 간접적으로 DNA에 직접 모집된다는 것을 발견했습니다. 최근 연구는 또한 CTCF, RNA 폴리머라제 II, 및 히스톤 마크의 게놈 전체 결합 위치를 프로파일라기 위해 ChIP-exo를 사용하여 인간 세포주18,19에서최신유전체 및 전사 메커니즘을 연구하였다.
사용할 수있는 ChIP 엑소 프로토콜의 여러 버전이 있습니다3,5,20. 그러나 이러한 ChIP 엑소 프로토콜은 차세대 시퀀싱 라이브러리 준비에 익숙하지 않은 연구를 위해 따르기 어렵다. ChIP 엑소 프로토콜의 우수한 버전은 따라하기 쉬운 지침과 비디오21로게시되었지만 완료하는 데 상당한 시간이 필요한 많은 효소 단계가 포함되어 있습니다. 여기서 는 감소된 효소 단계와 인큐베이션 시간을 포함하는 ChIP 엑소 프로토콜의 새 버전과 각 효소단계(21)에대한 설명을 보고합니다. 최종 수리 및 dA 꼬리 반응은 엔드 준비 효소를 사용하여 단일 단계로 결합됩니다. 인덱스 및 범용 어댑터 리칭 단계의 인큐베이션 시간은 리그레이션 인핸서를 사용하여 2시간에서 15분으로 단축됩니다. 이전 ChIP 엑소 프로토콜에 기재된 인덱스 어댑터 리그션 단계 후의 키나제 반응이 제거된다. 대신, 인산염 군은 오리고 DNA 합성 중에 인덱스 어댑터(표1)의5'끝 중 하나에 첨가되며, 이는 람다 엑소뉴클레아제 소화 단계에 사용될 것이다. 이전 ChIP 엑소 프로토콜은 RecJf exonuclease 소화를 사용하여 단일 좌초 DNA 오염 물질을 제거하는 동안, 이 소화 단계는 ChIP 엑소 라이브러리의 품질에 중요하지 않기 때문에 여기에서 제거됩니다. 또한, ChIP 용출 후 역방향 DNA를 정화하기 위해, 자성 비드 정제 방법은 페놀:클로로폼:이소아밀 알코올(PCIA) 추출 방법 대신에 사용된다. 이것은 DNA 추출의 잠복기 시간을 감소시킵니다. 중요한 것은 인덱스 어댑터 리그레이션 중에 형성된 대부분의 어댑터 디머를 제거하여 계각 매개 PCR의 효율성에 영향을 미칠 수 있습니다.
여기에 제시된 ChIP 엑소 프로토콜은 마우스 배아 줄기(ES) 세포와 분화된 포유류 뉴런에서 정밀한 단백질-DNA 상호작용의 검출에 최적화되어 있다. 간단히, 수확 및 교차 연결된 신경 세포는 크롬틴이 초음파 처리에 노출될 수 있도록, 다음 적절한 크기의 DNA 단편이 얻어지도록 초음파처리(도 1)를용해한다. 항체 코팅 구슬은 그 때 관심있는 단백질에 단편화된, 수용성 크로마틴을 선택적으로 면역침전염하는 데 사용됩니다. 면역 침전 DNA는 여전히 구슬에 있는 동안, 최종 수리, 시퀀싱 어댑터의 결찰, 채우기 반응 및 5' 3' 람다 exonuclease 소화 단계수행된다. 엑소뉴클레아제 소화 단계는 ChIP 엑소에게 초고해상도와 높은 신호 대 잡음 비율을 제공하는 것입니다. 람다 엑소뉴클레아제는 교차 연결 부위로부터 면역 침전 된 DNA를 몇 가지 염기 쌍 (bp)을 트림하여 오염 DNA가 저하됩니다. 엑소뉴클레아제 처리된 ChIP DNA는 항체 코팅 구슬로부터 용출되고, 단백질-DNA 크로스링크는 반전되고, 단백질은 저하된다. DNA는 단일 좌초 된 ChIP DNA에 추출되고 변성되고, 이중 좌초 DNA (dsDNA)를 만들기 위해 프라이머 어닐링 및 확장이 뒤따릅니다. 다음으로, 외핵체 처리 단부에 대한 범용 어댑터의 결찰이 수행된다. 생성된 DNA는 정제되고, PCR이 증폭되고, 겔정제되고, 차세대 시퀀싱을 실시한다.
ChIP 엑소 프로토콜은 ChIP-seq 프로토콜보다 길지만 기술적으로 는 그리 어렵지 않습니다. 모든 성공적으로 면역 침전된 ChIP DNA는 몇 가지 추가 효소 단계로 ChIP 엑소를 실시할 수 있습니다. 초고매핑 해상도, 배경 신호 감소, 게놈 결합 부위에 대한 거짓 긍정 및 네거티브 감소 등 ChIP-exo의 주목할 만한 장점은 시간 비용을 능가합니다.

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

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

게놈에 특정 단백질-DNA 상호 작용의 식별은 유전자 조절을 이해하는 데 중요합니다. 높은 처리량 시퀀싱 (ChIP-seq)과 결합된 크로마틴 면역 침전은 DNA 결합 단백질의 게놈 전체 결합 위치를 식별하는 데 널리 사용됩니다. 그러나 ChIP-seq 방법은 초음파 처리된 DNA 단편 및 비특이적 배경 DNA의 길이에 있는 이질성에 의해 제한되어 DNA 결합 부위의 낮은 매핑 해상도 및 불확실성을 초래합니다. 이러한 한계를 극복하기 위해 ChIP와 엑소뉴클레아제 소화(ChIP-exo)의 조합은 5'에서 3' 엑소뉴클레아제 소화를 활용하여 단백질-DNA 교차 링크 부위에 이질적으로 크기의 면역 침전DNA를 다듬는다. 엑소뉴클레아제 치료는 또한 비특이적 배경 DNA를 제거한다. 라이브러리가 준비된 외핵증 소화 DNA는 고처리량 시퀀싱을 위해 전송될 수 있다. ChIP 엑소 방법을 사용하면 감지 감도가 높아지고 배경 신호가 감소하는 근거리 쌍 매핑 해상도가 가능합니다. 포유류 세포 및 차세대 시퀀싱을 위한 최적화된 ChIP 엑소 프로토콜은 아래에 설명되어 있다.

도 2A는 마우스 ES 세포로부터 분화된 모터 뉴런 세포의 다양한 사이클을 통해 세포 리시스 및 초음파 처리 후 초음파 처리 결과를 도시한다. 초음파 처리 주기의 최적 수 (예를 들어, 도 2A에서12 사이클) 100-400 bp DNA 단편에서 강한 DNA 강도를 생성. 고품질 ChIP 엑소 라이브러리는 단편화된 크로마틴 DNA의 크기와 양을 기반으로 합니다. 따라서, ChIP 엑소를 ?...

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

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

게놈 전반에 걸친 단백질 결합 위치의 정확한 결정은 유전자 조절을 이해하는 데 중요합니다. 여기서는 외핵증 소화(ChIP-exo)를 사용한 크로마틴-면역침전DNA를 치료하는 게놈 매핑 방법을 설명하고 있으며, 이어서 고처리량 시퀀싱을 한다. 이 방법은 포유류 뉴런에서 근거리 쌍 매핑 해상도 및 높은 신호 대 잡음 비와 단백질 DNA 상호 작용을 검출합니다.

크롬 마틴 면역 침전 -엑소뉴클레아제 (ChIP-Exo)를 사용하여 마우스 줄기 세포 유래 뉴런에서 단백질 DNA 상호 작용의 고해상도 매핑

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

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

4.7K 조회수.  University of Toronto. 게놈 전반에 걸친 단백질 결합 위치의 정확한 결정은 유전자 조절을 이해하는 데 중요합니다. 여기서는 외핵증 소화(ChIP-exo)를 사용한 크로마틴-면역침전DNA를 치료하는 게놈 매핑 방법을 설명하고 있으며, 이어서 고처리량 시퀀싱을 한다. 이 방법은 포유류 뉴런에서 근거리 쌍 매핑 해상도 및 높은 신호 대 잡음 비와 단백질 DNA 상호 작용을 검출합니다.

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

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

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

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

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

ChEC-seq와의 단백질 - DNA 상호 작용의 게놈 전체 매핑<em&gt; 사카로 마이 세스 세레 비시 애</em

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

연구

유전학

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

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

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

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

마우스 T- 세포주에서 Chromatin Immunoprecipitation (ChIP)

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

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

네이티브 Chromatin Immunoprecipitation Murine 뇌 종양 Neurospheres를 사용 하 여

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

암 연구학

A Method to Study de novo Formation of Chromatin Domains

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity and/or disease. Despite tremendous efforts, our understanding of the formation and propagation of chromatin domains is still limited. Chromatin domains have been studied under steady-state conditions, which are not conducive to following the initial events during their establishment. Here, we present a method to inducibly reconstruct chromatin domains and follow their re-formation as a function of time. Although, first applied to the case of PRC2-mediated repressive chromatin domain formation, it could be easily adapted to other chromatin domains. The modification of and/or the combination of this method with genomics and imaging technologies will provide invaluable tools to study the establishment of chromatin domains in great detail. We believe that this method will revolutionize our understanding of how chromatin domains form and interact with each other.

A Method to Study de novo Formation of Chromatin Domains

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

크로마틴 도메인의 드 노보 형성을 연구하는 방법

The organization and structure of chromatin domains are unique to individual cell lineages. Their misregulation might lead to a loss in cellular identity ...

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

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, epigenetic regulators, and post-translation modifications of histones that combinatorially influence the gene expression state of nearby genes. RNA has also been shown to interact with various proteins to regulate chromatin dynamics&#160;and gene expression. Chromatin-associated RNA immunoprecipitation (CARIP) followed by next-generation sequencing (CARIP-Seq) is a novel method to survey RNAs associated with chromatin proteins, while chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) is a powerful genomics technique to map the location of post-translational modification of histones, transcription factors, and epigenetic modifiers on a global-scale in ES cells. Here, we describe methods to perform CARIP-Seq and ChIP-Seq, including library construction for next-generation sequencing, to generate global chromatin-associated RNA and epigenomic maps in ES cells.

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-Seq와 칩 Seq: Chromatin 관련 된 RNAs 및 배아 줄기 세포에 단백질 DNA 상호 작용을 식별 하는 방법

Embryonic stem (ES) cell self-renewal&#160;and differentiation is governed by extrinsic signals and intrinsic networks of transcription factors, ...

발생학

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

절연 및 신경 창시자 Chromatin-Immunoprecipitation 히스톤 3 리 79 Dimethylation 마크의 뒤의 재배

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

신경과학

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.

높은 처리량 시퀀싱을위한 고품질 염색질 면역 침전의 DNA 템플릿의 생성 (칩 SEQ)

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

생물학

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.

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.

칩 엑소 방법 : 가까운 자료 쌍의 정밀과 단백질 DNA 상호 작용을 확인

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

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

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: 셀 형식 관련 Epigenome 프로 파일링

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, and repair. Understanding the exact causality between genome topology and function is therefore crucial and increasingly the subject of intensive research. Chromosome conformation capture technologies (3C) allow inferring the 3D structure of chromatin by measuring the frequency of interactions between any region of the genome. Here we describe a fast and simple protocol to perform Capture Hi-C, a 3C-based target enrichment method that characterizes the allele-specific 3D organization of megabased-sized genomic targets at high-resolution. In Capture Hi-C, target regions are captured by an array of biotinylated probes before downstream high-throughput sequencing. Thus, higher resolution and allele-specificity are achieved while improving the time-effectiveness and affordability of the technology. To demonstrate its strengths, the Capture Hi-C protocol was applied to the mouse X-inactivation center (Xic), the master regulatory locus of X-chromosome inactivation (XCI).

Deciphering High-Resolution 3D Chromatin Organization via Capture Hi-C

This protocol describes the Capture Hi-C method used to characterize the 3D organization of megabased-sized targeted genomic regions at high-resolution, including boundaries of topologically associating domains (TADs) and long-range chromatin interactions between regulatory and other DNA sequence elements.

Capture Hi-C를 통한 고분해능 3D 염색질 조직 해독

The spatial organization of the genome contributes to its function and regulation in many contexts, including transcription, replication, recombination, ...