앨 러 배 마 건강 연구의 캐나다 학회에서 캐나다 대학원 장학금에 의해 지원 되었다. 이 작품은 게놈 브리티시 컬럼비아 및 캐나다 연구소의 건강 연구 (CIHR-120589) 캐나다 Epigenetics, 환경 및 건강 연구 컨소시엄 이니셔티브의 한 부분으로에서 교부 금에 의해 그리고 테리 폭스 연구 연구소 프로그램에 의해 지원 되었다 프로젝트 부여 #TFF-122869 수소와 수소에 테리 폭스 연구소 새로운 탐정 수상 (부여 # 1039)

<ol>
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저자 들은 아무 경쟁 금융 관심사 선언 합니다.

Chromatin 수정 및 transcriptional 규칙에서 위치 하는 nucleosome의 조합 성격을 감안할 때, 이러한 기능의 동시 측정 하는 방법 후 성적인 규정에 새로운 통찰력을 제공 것입니다. 여기에 제시 된 ndChIP-seq 프로토콜은 히스톤 수정 및 드문 1 차 셀9,10nucleosome 밀도 동시 심문 수 있도록 최적화 된 네이티브 칩 seq 프로토콜이입니다. ndChIP-seq 활용 chromatin의 효소 소화, 쌍 간 대규모 병렬 시퀀싱 및 가우스 혼합 배포 모델을 사용할 경우, 단일 nucleosome 수준 조사 히스톤 수정에 대 한 수 고는 deconvolution epigenomic 프로필 인구 내에서 성분에 의해 구동. 이 프로토콜을 사용 하 여, 우리 이전 면역을 시 켰 던 조각 크기, 쌍 간 경계, ChromHMM10,24에 의해 정의 된 특정 chromatin 상태와 관련 된 읽기에 의해 결정의 독특한 분포를 보고 있다.
NdChIP-seq 도서관의 질 항 체 특이성, 감도, 최적의 MNase 소화 조건, 및는 chromatin의 품질 등 여러 요인에 따라 달라 집니다. 사용 하는 항 체의 특이성은 성공적인 ndChIP-seq 라이브러리 생성에서 결정적 이다. 이상적인 항 체 다른 epitopes와 작은 교차 반응성으로 관심의 피토 프에 대 한 높은 선호도 보여줍니다. 그것은 동등 하 게 자석 구슬 선택의 항 체에 대 한 높은 선호도 선택 하는 것이 중요입니다.
MNase 소화가이 프로토콜에 중요 하 고 시간 및 농도 민감한 반응입니다. 따라서, 여러 개의 샘플을 처리할 때 그것은 중요 한 시간의 상응 하는 금액에 대 한 각 반응에 대 한 알을 품는 (단계 2.2 참조). chromatin의 품질이 크게 ndChIP이 Fragmented chromatin 리드 낮은 신호 대 잡음 비와 최적의 MNase 소화 프로필 및 라이브러리에서 결과의 결과 초래 하는 또 다른 요인은. 낮은 기본 샘플 세포 생존 능력 또는 염색 질, 타락 한 같은 포 르 말린 고정 파라핀 포함 (FFPE) 조직이이 프로토콜에 대 한 권장 하지 않습니다.
Chromatin 추출 하는 동안 그림의 추가 감소 (즉, 무작위) chromatin 조각화 및 보존의 무결성 히스톤 꼬리를 바라지 않는. 따라서, 그림 사용 하 여 세포의 용 해 버퍼와 그냥 이전 immunoprecipitation 버퍼에 추가 해야 합니다. Cytometry 통해 선택 셀 가능한 셀을 선택 하 고 확인 하는 동안 셀 셀 번호 추정의 정확성과 세포의 생존 능력을 증가 하는 낮은 흐름 율에 정렬 됩니다. 세포 세포의 용 해 버퍼에 직접 정렬 하지 마십시오. 칼 집 버퍼 세포의 용 해 버퍼를 희석 하 고 세포 막의 효과적인 permeabilization MNase를 방지. 셀 또는 유기 체의 종류에 따라 적정 MNase의 최적의 소화를 얻기 위해 필요할 수 있습니다.
ndChIP-seq 포유류 세포에 광범위 한 표시 및 입력에 대 한 좁은 표시 및 100 백만 쌍 읽기 (50 백만 조각) 50 백만 쌍 읽기 (25 백만 조각)의 최소 시퀀싱 깊이 필요합니다. 가우스 혼합물 분배 알고리즘 라이브러리가이 추천 아래 크게 깊이 시퀀스에 최적으로 수행 되지 않습니다. ndChIP-seq는 모노 또는 디 nucleosome 지배 발기인으로 모노 및 디 nucleosome 조각 길이 대 한 가중치 분포 값 간의 구분이 약간 발기인 분류 하지 것입니다. 따라서, 이러한 발기인 이후 분석에서 제거 되어야 합니다. 생물 복제 예측된 배포판에 대 한 자신감을 증가 하 고 MNase 소화 및 도서관 건설에서 기술 변화를 식별을 생성할 수 있습니다.
네이티브 칩 seq 프로토콜의 이전 반복, 달리 ndChIP-seq nucleosome 밀도, 통합 조각 크기 게시물 immunoprecipitation를 이용 하 여 염색 질 구조와 히스톤 수정의 조합 효과 조사 하는 수단 제공 MNase 접근성, 히스톤 수정 측정에 의해 결정. NdChIP-seq의 응용 프로그램 기본 세포와 조직에 epigenetic 규제의 통합 특성에 새로운 통찰력을 제공 하 고 인구 내의 성분으로 인해 후 서명 식별을 허용.

진 핵 게놈 DNA1,2의 146의 기본적인 쌍에 의해 circumscribed 4 개의 히스톤 단백질 (예를 들어, H2A, H2B, H3, H4)의 두 개의 복사본으로 구성 하는 nucleosome 구조 반복을 통해 chromatin에 패키지 됩니다. Chromatin 개장 단지 유전자 발기인 경계 내 nucleosome 위치 제어 및 유전자 발현의 규칙에 참여 하는 전사 인자와 RNA 중 합 효소 기계3, DNA의 액세스 가능성을 변경 하 여 4.
nucleosome 내 히스톤의 아미노 맨끝 꼬리 acetylation, 메 틸 화, 인 산화, ubiquitylation, sumoylation, formylation, 및 특정 아미노산5 의 hydroxylation를 포함 하 여 다양 한 화학식 수정 대상이 되는 , 6 , 7 , 8. 위치 및 이러한 변경의 영향 전사7의 정품 인증을 허용 하는 분자 복합물의 염색 질 구조 및 제어 액세스 chromatin 상태 지시. 그 두 nucleosome 밀도 히스톤 수정 유전자 전사의 로컬 컨트롤에서 역할을 할 우리 네이티브 칩 접근 nucleosome 밀도 히스톤 수정9의 동시 측정을 가능 하 게 개발 10.
기본 칩 seq endonuclease micrococci nuclease 핵11,12, nucleosome13 위치를 지도에 활용 되는 속성 내에서 기본 상태에서 그대로 chromatin 소화 (MNase)의 활용 , 14 , 15. nucleosome 밀도 칩 seq (ndChIP-seq) 히스톤 수정10MNase 접근을 결합 하는 측정을 생성 하는 chromatin의 영역을 열려면 MNase의 특혜 접근의 속성 활용. ndChIP-seq는 히스톤 수정 드문 1 차 셀, 조직 및 배양된 세포에서의 프로 파일링 적합 합니다. 여기, 선물이 적합 조각 크기 게시물 immunoprecipitation, 쌍 간 경계, 읽기에 의해 결정을 통합 하는 앞에서 설명한 분석 프레임 작업10 시퀀스 데이터 집합 생성을 가능 하 게 상세한 프로토콜 동시에 히스톤 수정 측정 MNase 접근성 조사. 이전에, 10000 기본 인간 코드 혈액이 프로토콜의 응용 프로그램 파생 CD34+ 세포와 인간 배아 줄기 세포 염색 질 구조와 히스톤 수정이 내 간의 독특한 관계 셀 유형10 공개 . NdChIP-seq 단일 nucleosome 수준에서 세포 인구에서 epigenomic 기능을 공개 하 고 다른 유형의 서명으로 해결은 nucleosome 접근성 및 히스톤 수정 동시에 측정 하는 능력을 감안할 때, 그들의 구성 요소입니다. NdChIP-seq에 의해 다른 유형의 세포 인구의 탐험의 예는 어디 H3K4me3, 적극적인 마크와 H3K27me3, 억압 적인 마크는 현재10가 발기인의 조사입니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	
	<tbody>
		<tr >
			<td >1M Tris-HCl &#8211;pH 7.5</td>
			<td >Thermo Scientific</td>
			<td >15567-027</td>
			<td ></td>
		</tr>
		<tr >
			<td >1M Tris-HCl &#8211;pH 8</td>
			<td >Thermo Scientific</td>
			<td >15568-025</td>
			<td></td>
		</tr>
		<tr >
			<td >0.5M EDTA</td>
			<td >Thermo Scientific</td>
			<td >AM9260G</td>
			<td></td>
		</tr>
		<tr >
			<td >5M NaCl</td>
			<td >Sigma</td>
			<td >1001385276</td>
			<td></td>
		</tr>
		<tr >
			<td >Triton X-100</td>
			<td >Sigma</td>
			<td >1001412354</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium-Deoxycholate</td>
			<td >Sigma</td>
			<td >1001437582</td>
			<td></td>
		</tr>
		<tr >
			<td >SDS</td>
			<td >Thermo Scientific</td>
			<td >15525-017</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium-Bicarbonate</td>
			<td >Fisher Scientific</td>
			<td >S233-500</td>
			<td></td>
		</tr>
		<tr >
			<td >100% EtOH</td>
			<td >NA</td>
			<td >NA</td>
			<td></td>
		</tr>
		<tr >
			<td >V-bottom 96 well plate (AB 1400)</td>
			<td >Thermo Scientific</td>
			<td >AB-1400-L</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P2 pipetman</td>
			<td >Mandel</td>
			<td >F144801</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P10 pipetman</td>
			<td >Mandel</td>
			<td >F144802</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P20 pipetman</td>
			<td >Mandel</td>
			<td >F123600</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P100 pipetman</td>
			<td >Mandel</td>
			<td >F123615</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P200 pipetman</td>
			<td >Mandel</td>
			<td >F123601</td>
			<td></td>
		</tr>
		<tr >
			<td >Gilson P1000 pipetman</td>
			<td >Mandel</td>
			<td >F123603</td>
			<td></td>
		</tr>
		<tr >
			<td >Micrococcal Nuclease&#160;</td>
			<td >NEB</td>
			<td >M0247S</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo Mixer C (Heating block mixer)</td>
			<td >Eppendorf</td>
			<td >5382000023</td>
			<td></td>
		</tr>
		<tr >
			<td >Multi 12-channel Pippet P20</td>
			<td >Rainin</td>
			<td >17013803</td>
			<td></td>
		</tr>
		<tr >
			<td >Multi 12-channel Pippet P200</td>
			<td >Rainin</td>
			<td >17013805</td>
			<td></td>
		</tr>
		<tr >
			<td >1.5 ml LoBind tube</td>
			<td >Eppendorf</td>
			<td >22431021</td>
			<td></td>
		</tr>
		<tr >
			<td >0.5 ml LoBind tube</td>
			<td >Eppendorf</td>
			<td >22431005</td>
			<td></td>
		</tr>
		<tr >
			<td >Plastic Plate Cover</td>
			<td >Edge Bio</td>
			<td >48461</td>
			<td></td>
		</tr>
		<tr >
			<td >PCR cover</td>
			<td >Bio Rad</td>
			<td >MSD-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Aluminum Plate Cover</td>
			<td >Bio Rad</td>
			<td >MSF-1001</td>
			<td></td>
		</tr>
		<tr >
			<td >Elution buffer (EB buffer)</td>
			<td >Qiagen</td>
			<td >19086</td>
			<td></td>
		</tr>
		<tr >
			<td >1M DTT</td>
			<td >Sigma</td>
			<td >646563</td>
			<td></td>
		</tr>
		<tr >
			<td >Eppendorf 5810R centrifuge&#160;</td>
			<td >Eppendorf</td>
			<td >22625004</td>
			<td></td>
		</tr>
		<tr >
			<td >Ultrapure water</td>
			<td >Thermo Scientific</td>
			<td >10977-015</td>
			<td></td>
		</tr>
		<tr >
			<td >Protein G dynabeads</td>
			<td >Thermo Scientific</td>
			<td >10001D</td>
			<td></td>
		</tr>
		<tr >
			<td >Protein A dynabeads</td>
			<td >Thermo Scientific</td>
			<td >10001D</td>
			<td></td>
		</tr>
		<tr >
			<td >Protease inhibitor Cocktail</td>
			<td >Calbiochem</td>
			<td >539134</td>
			<td></td>
		</tr>
		<tr >
			<td >Rotating platform</td>
			<td >Mandel</td>
			<td >1b109-12052010</td>
			<td></td>
		</tr>
		<tr >
			<td >Plate magnet</td>
			<td >Alpaqua</td>
			<td >2523</td>
			<td></td>
		</tr>
		<tr >
			<td >Domed 12-cap strip</td>
			<td >Bio Rad</td>
			<td >TCS1201</td>
			<td></td>
		</tr>
		<tr >
			<td >Buffer G2</td>
			<td >Qiagen</td>
			<td >1014636</td>
			<td></td>
		</tr>
		<tr >
			<td >Protease</td>
			<td >Qiagen</td>
			<td >19155</td>
			<td></td>
		</tr>
		<tr >
			<td>Tube Magnet (DynaMag-2)</td>
			<td >Thermo Scientific</td>
			<td >12321D</td>
			<td></td>
		</tr>
		<tr >
		<td>Tube Magnet (DynaMag-2)</td>
			<td >LabCore</td>
			<td >730-006</td>
			<td></td>
		</tr>
		<tr >
			<td >V shape Plastic reservoir</td>
			<td >Mandel</td>
			<td >S0100A</td>
			<td></td>
		</tr>
		<tr >
			<td >Vortex</td>
			<td >Mandel</td>
			<td >C1801</td>
			<td></td>
		</tr>
		<tr >
			<td >Mini Fuge</td>
			<td >Mettler Toledo</td>
			<td >XS2035</td>
			<td></td>
		</tr>
		<tr >
			<td >Analytical scale</td>
			<td >Mandel</td>
			<td >LB109-12052010</td>
			<td></td>
		</tr>
		<tr >
			<td >Quick Ligation Reaction Buffer, 5X</td>
			<td >NEB</td>
			<td>B6058S</td>
			<td></td>
		</tr>
		<tr >
			<td >DNA Polymerase I, Large (Klenow) Fragment</td>
			<td >NEB</td>
			<td>M0210S</td>
			<td></td>
		</tr>
		<tr >
			<td >Klenow Fragment (3&#39;-5&#39; exo)</td>
			<td >NEB</td>
			<td>M0212S</td>
			<td></td>
		</tr>
		<tr >
			<td >T4 DNA Polymerase</td>
			<td >NEB</td>
			<td>M0203S</td>
			<td></td>
		</tr>
		<tr >
			<td >T4 Polynucleotide Kinase</td>
			<td >NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr >
			<td >Deoxynucleotide Solution mix, 10mM</td>
			<td >NEB</td>
			<td>N0447S</td>
			<td></td>
		</tr>
		<tr >
			<td >dATP Solution 10mM</td>
			<td >NEB</td>
			<td>N0440S</td>
			<td></td>
		</tr>
		<tr >
			<td >Quick T4 DNA Ligase</td>
			<td >NEB</td>
			<td>M0202T</td>
			<td></td>
		</tr>
		<tr >
			<td >Adenosine 5&#39;-Triphosphate (ATP), 25mM</td>
			<td >NEB</td>
			<td>P0756S</td>
			<td></td>
		</tr>
		<tr >
			<td >DNA Polymerase</td>
			<td >Thermo Scientific</td>
			<td>F549L</td>
			<td></td>
		</tr>
		<tr >
			<td >NEBuffer 2</td>
			<td >NEB</td>
			<td>B7002S</td>
			<td></td>
		</tr>
		<tr >
			<td >Hank&#39;s buffered salt solution</td>
			<td >Thermo Scientific</td>
			<td>14025076</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal bovine serum&#160;</td>
			<td >Thermo Scientific</td>
			<td>A3160702</td>
			<td></td>
		</tr>
		<tr >
			<td >phosphate buffer saline&#160;</td>
			<td >Thermo Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K4me3</td>
			<td >Cell Signaling</td>
			<td >9751S</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K4me1</td>
			<td >Diagenode</td>
			<td >C15410037</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K27me3</td>
			<td >Diagenode</td>
			<td >pAb-069-050</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K36me3</td>
			<td >Abcam</td>
			<td >ab9050</td>
			<td></td>
		</tr>
		<tr >
			<td >H3K9me3</td>
			<td >Diagenode</td>
			<td >C15410056</td>
			<td></td>
		</tr>
		<tr >
			<td >SeraMag&#160; super-paramagnetic beads</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of native chromatin immunoprecipitation sequencing libraries for nucleosome density analysis

선물이 시퀀싱 (칩 seq) 실험 프로토콜 호환 가우스 혼합물 배급 기반 분석 방법론 (nucleosome 밀도 칩 seq; ndChIP-seq)의 생성을 가능 하 게 수정 된 네이티브 chromatin immunoprecipitation 히스톤 수정 게놈 넓은 micrococcal nuclease (MNase) 접근의 결합 된 측량. Nucleosome 위치 및 로컬 밀도, 그리고 그들의 히스톤 subunits posttranslational 수정 로컬 전송 상태를 조절 하는 콘서트에서 행동. NdChIP-seq에 의해 생성 된 히스톤 수정 nucleosome 접근의 조합 측정 이러한 기능의 동시 심문 수 있습니다. NdChIP-seq 방법론 1 차 셀 기반된 칩 seq 프로토콜 cross-linking에 액세스할 수의 작은 숫자에 적용 됩니다. 함께 찍은, ndChIP-seq 드문 1 차 셀 인구 내에서 RNA 전사 조절 공유 메커니즘에 새로운 통찰력을 얻기 위해 로컬 nucleosome 밀도와 히스톤 수정 조합에서의 측정을 수 있습니다.

Watch this Scientific Journal Video about Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis at JoVE.com

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

네이티브 Chromatin Immunoprecipitation Nucleosome 밀도 분석을 위한 라이브러리를 시퀀싱의 세대

선물이 nucleosome 밀도 칩 seq 분석 프레임 워크를 통합 하는 micrococcal nuclease (MNase) 접근에 대 한 적합 한 시퀀스 데이터 집합의 세대에 대 한 (칩 seq) 방법론을 시퀀싱 수정된 네이티브 chromatin immunoprecipitation 히스톤 수정 측량와.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

generation-native-chromatin-immunoprecipitation-sequencing-libraries

Research

JoVE Journal

Genetics

22.0K 조회수.  University of British Columbia. 선물이 nucleosome 밀도 칩 seq 분석 프레임 워크를 통합 하는 micrococcal nuclease (MNase) 접근에 대 한 적합 한 시퀀스 데이터 집합의 세대에 대 한 (칩 seq) 방법론을 시퀀싱 수정된 네이티브 chromatin immunoprecipitation 히스톤 수정 측량와.

비디오: Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

바인딩 속성 수정 복합물 Chromatin의 대량 Chromatin의 분석에 대 한 순차적 소금 기사

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous ...

연구

유전학

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

차세대 시퀀싱에 적합한 식물성 세포질 DNA 농축 방법의 최적화 및 비교 분석

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Chromatin Immunoprecipitation을 이용한 c-KIT 리간드 프로모터 분석

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

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 ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

낮은 풍부 배아 샘플 chromatin Immunoprecipitation (칩) 프로토콜

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

단단히 준비 초기 초파리 배아에서 게놈 넓은 Chromatin 구조 캡처 라이브러리의 생성

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

히스톤 수정 Saccharomyces cerevisiae 에서 chromatin Immunoprecipitation (칩)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Murine 갈색 지방이 많은 직물의 염색 질 Immunoprecipitation

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene expression. The cellular transcriptional machinery and its chromatin modification proteins coordinate to regulate transcription. To analyze transcriptional regulation at the molecular level, several experimental methods such as electrophoretic mobility shift, transient reporter and chromatin immunoprecipitation (ChIP) assays are available. We describe a modified ChIP assay in detail in this article because of its advantages in directly showing histone modifications and the interactions between proteins and DNA in cells. One of the key steps in a successful ChIP assay is chromatin shearing. Although sonication is commonly used for shearing chromatin, it is difficult to identify reproducible conditions. Instead of shearing chromatin by sonication, we utilized enzymatic digestion with micrococcal nuclease (MNase) to obtain more reproducible results. In this article, we provide a straightforward ChIP assay protocol using MNase.

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

포유류 세포에서 미세 Coccal 뉴클레아제사용 염색질 면역침전 분석

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA의 발자국 의 식별:연속RNA 면역 침전을 통한 단백질 복합체 시퀀싱 (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...