Name: high-resolution mapping of protein-dna interactions in mouse stem cell-derived neurons using chromatin immunoprecipitation-exonuclease (chip-exo)
Uploaded: 2020-08-14T16:00:00
Description: 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

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.

참고: 오토클레이브 증류및 탈이온수(ddH2O)는 버퍼 및 반응 마스터 믹스를 만드는 데 권장됩니다. 섹션 1-4세포 용해 및 초음파 처리를 설명하고, 섹션 5-7은 크로마티닌 면역 침전 (ChIP),섹션 8-11구슬에 대한 효소 반응을 설명하고, 섹션 12 및 13은 ChIP 용출 및 DNA 정화를 설명하고, 섹션 14-19는 도서관 준비를 설명합니다.
1. 셀 을 수확하고 교차 연결
<ol><li>마?...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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&#160;</td>
			<td >BioShop</td>
			<td >HEP003</td>
			<td >Lysis Buffer 1, High Salt Wash Buffer</td>
		</tr>
		<tr >
			<td >Klenow Fragment (3-&#62;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&#160;</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, &#8805;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&#160;</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&#160;</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&#160;</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&#160;</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 -&#62; 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 ...

The main advantage of our ChIP-exo method is the improved mapping resolution of protein DNA interactions in the cell compared to the conventional chromatin immunoprecipitation method. To prepare protein G magnetic beads for ChIP, mix the magnetic beads until homogeneous. Then add 25 microliters of the magnetic beads to a two milliliter protein low bind tube.Add one milliliter of blocking solution to the beads. Mix well by pipetting, then place the tube on a magnetic rack for one minute. Once the supernatant is clear, remove it.Next, add one milliliter of blocking solution to the beads. Place the tube on a rocking platform at four degrees Celsius and rock for 10 minutes, then briefly spin the tube. Place the tube on the magnetic rack and remove the supernatant.Add 500 microliters of blocking solution to the magnetic beads. Briefly spin the antibody against Islet-1, then add four micrograms of the antibody to the tube containing the magnetic beads. Place the tube containing magnetic beads and Islet-1 antibody on a rocking platform at four degrees Celsius and rock for six to 24 hours.To begin the chromatin immunoprecipitation, wash the antibody-coated beads in one milliliter of blocking solution. Place the tube containing the antibody-coated beads on a rocking platform at four degrees Celsius for five minutes. After briefly spinning, place the tube on a magnetic rack and remove the supernatant.Resuspend the antibody-coated beads in 50 microliters of blocking solution. Add one milliliter of sonicated lysates to the antibody-coated beads, then incubate the sample on a rocking platform at four degrees Celsius overnight. After allowing the sample to incubate overnight, collect liquid from the cap by briefly spinning the tube, then place the tube on a magnetic rack.And after one minute, remove the supernatant carefully with a pipette. Next, perform a series of washes as follows. Add one milliliter of cold wash buffer containing CPI to the sample.Mix the sample on a rocking platform at four degrees Celsius for five minutes. Briefly spin the sample tube, then place on a magnetic rack. After one minute, remove the supernatant with a pipette.For the first enzymatic reaction and repair and DA tailing, add 38 microliters of autoclaved double distilled water to the sample, then add end prep reaction mix and end prep enzyme mix. Mix the contents of the tube by pipetting, then incubate the sample at 20 degrees Celsius for 30 minutes. Next, wash the sample beads with high salt wash buffer, lithium chloride wash buffer, and 10 millimolar Tris hydrogen chloride buffer as described previously.To perform the index adapter ligation, add 27 microliters of cold 10 millimolar Tris hydrogen chloride buffer to the sample. Then add index adapter, ligation enhancer, and ligase master mix. Incubate the sample at 20 degrees Celsius for 15 minutes.Again, wash the sample beads with high salt wash buffer, lithium chloride wash buffer, and 10 millimolar Tris hydrogen chloride buffer. Then add 47 microliters of cold 10 millimolar Tris hydrogen chloride buffer to the sample. To repair the nick in the DNA resulting from the missing phosphodiester bond, add 11.1 microliters of the fill-in mix to the sample.Incubate the sample at 30 degrees Celsius for 20 minutes. After a series of washes, add 50 microliters of cold autoclaved double distilled water to the sample. To digest the ChIP DNA in the five prime to three prime direction, add lambda exonuclease buffer and lambda exonuclease and mix the sample by pipetting.Incubate the sample at 37 degrees Celsius for 30 minutes. After the digestion is complete, repeat the three washes a final time. To elute ChIP sample from the beads, resuspend the sample in 75 microliters of ChIP elution buffer and incubator at 65 degrees Celsius for 15 minutes at 130 times G.Add 2.5 microliters of 20 milligrams per milliliter proteinase K to the sample.Vortex the sample briefly, then incubate overnight at 65 degrees Celsius. Briefly spin the sample and place on a magnetic rack. After one minute, transfer the supernatant to a new 1.5 milliliter tube.Purify and elute the DNA. After purifying the eluted DNA, transfer 16 microliters of the extracted DNA sample to a PCR tube. Add 1.2 microliters of denaturing and primer annealing mix to the sample.Using the program described in table six of the manuscript, denature and anneal primers to the template DNA. Add three microliters of primer extension mix to the sample and run the PCR program described in table seven of the manuscript. Add 4.1 microliters of DA tailing mix to the sample and run the sample using the PCR program in table eight of the manuscript.Then add 21.5 microliters of universal adapter ligation mix to the sample and incubate for 15 minutes at 20 degrees Celsius. Add 29 microliters of LM-PCR mix to the sample, then run the sample using the program from table 10 of the manuscript. Following 18 cycles of LM-PCR, ChIP-exo samples from mouse motor neurons were electrophoresed on a 1.5%agarose gel.The results for ChIP-exo Islet-1 showed amplified DNA libraries around 200 to 400 base pairs indicating that the ChIP-exo libraries were successfully amplified by LM-PCR. The band around 100 base pairs represents artifacts from adapters and PCR primers. The no antibody control demonstrates that non-specific background DNA was digested by the lambda exonuclease treatment.Using ChIP-exo and ChIP-seq, Islet-1 bound locations were identified. The ChIP-exo signal was highly focused at Islet-1 binding sites detecting multiple clustered Islet-1 transcription factor binding patterns. The ChIP-seq signal displayed broader signals indicating that ChIP-exo had higher mapping resolution than ChIP-seq for Islet-1.Multiple transcription factors often bind DNA next to each other as a protein-DNA complex. ChIP-exo allows us to study how multiple transcription factors recognize and regulate target DNA.

Research

JoVE Journal

Genetics

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

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