Name: the chip-exo method: identifying protein-dna interactions with near base pair precision
Uploaded: 2016-12-23T15:00:00
Description: Watch this Scientific Journal Video about The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision at JoVE.com

We thank the Venters Lab and members of the Molecular Physiology and Biophysics Department for helpful discussions. Special thanks to Frank Pugh for his guidance, mentoring, and many insightful discussions on the nuances of ChIP-exo while I was a post-doctoral fellow in his laboratory.

참고 : 두 번 증류수 H 2 O 또는 분자 수준의 상응하는 모든 버퍼와 반응 믹스하는 것이 좋습니다. 날 0 : 재료 준비 및 세포 수확 1. 버퍼 준비 <ol><li> 3 (표 1 - - 3) 용해 버퍼 (1)을 준비하고 사용하기 직전에 각 버퍼의 50ml로 100 ㎕의 완전한 단백질 분해 효소 억제제 재고 (CPI)를 추가합니다. 1 정 1 ml의를 분자 수준의 H 2</s...

<ol>
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<html> 우리는 염색질 근처 염기쌍 해상도 편견, 게놈 차원의 방식으로 단백질 상호 작용에 대한 정확한 결합 위치를 결정하기 위해 기능 유전체학 프로토콜을 제시한다. 면역 침전이 자성 수지에 남아있는 동안 근처 염기쌍 매핑 해상도를 얻기 위해 가장 중요한 단계는 칩 - 풍부 DNA의 엑소 뉴 클레아 제 처리이다. 표면 상, 단백질 복합체는 잠재적으로 주어진 서브 유닛 (예를 들어, 크로 마?...

크로 마틴 면역 침강 (칩)을 선택적으로 살아있는 세포에서 특정 단백질과 상호 작용하는 DNA 단편을 위해 농축에 의해 유전자 조절 메커니즘을 연구하는 강력한 방법이다. 기술은 높은 처리량 시퀀싱 (칩-SEQ) 1 올리고 뉴클레오티드 마이크로 어레이 (칩 - 칩)에 하이브리드에 하나의 궤적 (표준 칩-qPCR에) 검출에서 향상으로 칩 농축 DNA 단편의 검출 방법은 진화했다. 칩-서열이 광범위한 응용 프로그램, 염색질 이질성과 비특이적 DNA 상호 작용을 볼 수 있지만 오탐 (false positive)과 부정확 한 매핑 최고의 데이터 품질을 방해했다. 이러한 제한을 우회하기 위해, 박사 프랭크 퓨는 칩 - 엑소 방법 (2)를 개발했다. 칩 엑소의 두드러진 특징은 효율적으로 위치를 결합 전사 인자를 footprinting에, 엑소 뉴 클레아 &#39;3&#39;, 5를 포함하고 있다는 것이다. 결과적으로, 칩 - 엑소 방법은 높은 해상도 detectio의 큰 동적 범위를 달성n 및 낮은 소음. 칩 - 엑소 좀 더 기술적으로 어려운 칩-서열보다 마스터하지만 연구는 다양한 생물학적 시스템 3-8를 사용하여 고유의 초 고해상도 통찰력을 확보하는 것을 목표로, 그것은 널리 채택되고있다. 실제로, 칩 엑소 성공적 박테리아, 효모, 마우스, 래트, 인간 세포 시스템에 적용되어왔다. 원리의 증거로, 칩 - 엑소는 원래 효모 전사의 소수는 2 요인에 대한 정확한 바인딩 모티브를 식별하는 데 사용되었다. 이 기술은 또한 전사 개시전 복합체의 구성을 연구하기 위해 다양한 히스톤 9,10 subnucleosomal의 구조를 해독 효모에 사용 하였다. 최근에, 우리는 인간의 발기인에 이벤트를 바인딩 인접 TFIIB와 폴 II를 해결하기 위해 칩 - 엑소을 활용, 별개의 개시 11 복합체에서 널리 발산 전사가 발생하는 것으로 나타났다. 여기에 제시된 워크 플로우 최적화의있다포유 동물 칩 - 엑소 (그림 1)에 대한 treamlined. 첫째, 생활 차 또는 조직 배양 세포를 공유 가교를 통해 생체 내 단백질 DNA 상호 작용에 보존하는 포름 알데히드로 처리됩니다. 세포 용균 및 염색질은 100 ~ 전단된다 - 500 염기쌍 크기 단편. 칩은 선택적으로 관심있는 단백질에 가교 결합 된 DNA 단편에 대한 풍부. 이 시점에서, 칩은 일반적 서열 라이브러리 본질적 수백 염기쌍 단편의 평균 크기를 검출 해상도를 제한하는 제조된다. 그러나, 칩 엑소 왼쪽 및 람다 엑소 뉴 클레아 제와 단백질의 DNA 가교 부위의 바로 5 &#39;DNA 경계를 트리밍함으로써 이러한 제한을 극복한다. 아래에 설명 된대로 시퀀싱 라이브러리는 다음 엑소 뉴 클레아 제 소화 DNA로 구성되어있다. 얻어진 중첩 5 &#39;테두리 단백질 DNA 상호 작용 (도 1, 스텝 14)의 생체 내 공간을 나타내고, 높은 처리량 시퀀싱에 의해 검출된다. Alt 키무릎이 칩 엑소 방법은 칩-서열보다 더 복잡 대부분의 단계 사이의 전환은 샘플 손실과 실험적인 변동성을 최소화 간단한 비드 세척을 필요로한다. 중요한 것은, 칩 - 엑소는 칩-SEQ, 또한 칩 - 엑소에 성공한다 칩-서열에 성공하는 샘플의 세련된 버전 때문이다. 칩 서열에서 근본적으로 구별되는 데이터 구조의 칩 엑소 결과 단백질 DNA의 상호 작용의 생체 배출량 측정. 공통 칩 서열 발신자가 가장 정확한 피크 호출을 수득 칩 엑소 데이터에 적용될 수 있지만, 우리는 특별히 염두 칩 엑소 데이터의 독특한 구조로 설계 정보학 도구를 추천한다. 이들은 Genetrack, GEM, MACE, Peakzilla 및 ExoProfiler 12-15을 포함한다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >37% formaldehyde, methanol free, 10x10ml ampules</td>
			<td >ThermoFisher Scientific</td>
			<td >28908</td>
			<td >Section 3</td>
		</tr>
		<tr >
			<td >Complete Protease Inhibitor cocktail (CPI)</td>
			<td >Roche Life Science</td>
			<td >11873580001</td>
			<td >Sections 4, 8</td>
		</tr>
		<tr >
			<td >Bioruptor sonicator</td>
			<td >Diagenode</td>
			<td >UCD200</td>
			<td >Section 5</td>
		</tr>
		<tr >
			<td >15 ml polystyrene tubes</td>
			<td >BD Falcon</td>
			<td >352095</td>
			<td >Section 5</td>
		</tr>
		<tr >
			<td >MagSepharose Protein G Xtra beads</td>
			<td >GE Healthcare</td>
			<td >28-9670-66</td>
			<td >Section 6</td>
		</tr>
		<tr >
			<td >DynaMag-1.5ml Side Magnetic Rack</td>
			<td >Invitrogen</td>
			<td >12321D</td>
			<td >Sections 6-17, 22</td>
		</tr>
		<tr >
			<td >Mini-Tube Rotator</td>
			<td >Fisher Scientific</td>
			<td >05-450-127</td>
			<td >Sections 7, 22</td>
		</tr>
		<tr >
			<td >T4 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0203</td>
			<td >Section 9</td>
		</tr>
		<tr >
			<td >DNA Polymerase I, Klenow</td>
			<td >New England BioLabs</td>
			<td >M0210</td>
			<td >Section 9</td>
		</tr>
		<tr >
			<td >10x NEBuffer 2 (10x Reaction Buffer 2)</td>
			<td >New England BioLabs</td>
			<td >B7002</td>
			<td >Sections 9-11, 13, 15, 20</td>
		</tr>
		<tr >
			<td >Thermomixer C</td>
			<td >Eppendorf</td>
			<td >5382</td>
			<td >Sections 9-16</td>
		</tr>
		<tr >
			<td >ATP</td>
			<td >Roche Life Science</td>
			<td >010419979001</td>
			<td >Sections 9, 11, 13</td>
		</tr>
		<tr >
			<td >dNTPs</td>
			<td >New England BioLabs</td>
			<td >N0447</td>
			<td >Sections 9, 12, 19, 23</td>
		</tr>
		<tr >
			<td >T4 Polynucleotide Kinase</td>
			<td >New England BioLabs</td>
			<td >M0201</td>
			<td >Sections 9, 13</td>
		</tr>
		<tr >
			<td >dATP</td>
			<td >New England BioLabs</td>
			<td >N0440</td>
			<td >Sections 10, 20</td>
		</tr>
		<tr >
			<td >Klenow 3&#39;-5&#39; Exo Minus</td>
			<td >New England BioLabs</td>
			<td >M0212</td>
			<td >Sections 10, 20</td>
		</tr>
		<tr >
			<td >T4 DNA ligase&#160;</td>
			<td >New England BioLabs</td>
			<td >M0202</td>
			<td >Sections 11, 21</td>
		</tr>
		<tr >
			<td >&#934;-29 DNA Polymerase</td>
			<td >New England BioLabs</td>
			<td >M0269</td>
			<td >Sections 12, 19</td>
		</tr>
		<tr >
			<td >10x &#934;-29 Buffer</td>
			<td >New England BioLabs</td>
			<td >B0269</td>
			<td >Sections 12, 19</td>
		</tr>
		<tr >
			<td >Lambda Exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0262</td>
			<td >Section 14</td>
		</tr>
		<tr >
			<td >10x Lambda Buffer</td>
			<td >New England BioLabs</td>
			<td >B0262</td>
			<td >Section 14</td>
		</tr>
		<tr >
			<td >RecJf Exonuclease</td>
			<td >New England BioLabs</td>
			<td >M0264</td>
			<td >Section 15</td>
		</tr>
		<tr >
			<td >Proteinase K</td>
			<td >Roche Life Science</td>
			<td >03115828001</td>
			<td >Section 16</td>
		</tr>
		<tr >
			<td >Glycogen</td>
			<td >Roche Life Science</td>
			<td >010901393001</td>
			<td >Section 18</td>
		</tr>
		<tr >
			<td >10x T4 Ligase Buffer</td>
			<td >New England BioLabs</td>
			<td >B0202</td>
			<td >Section 21</td>
		</tr>
		<tr >
			<td >AMPure XP (clean up) beads&#160;</td>
			<td >Beckman Coulter</td>
			<td >A63881</td>
			<td >Section 22</td>
		</tr>
		<tr >
			<td >Q5 Hot Start DNA Polymerase&#160;</td>
			<td >New England BioLabs</td>
			<td >M0493</td>
			<td >Section 23</td>
		</tr>
		<tr >
			<td >5x Q5 Buffer (5x PCR Buffer)</td>
			<td >New England BioLabs</td>
			<td >B9027</td>
			<td >Section 23</td>
		</tr>
		<tr >
			<td >Qubit Fluorometer</td>
			<td >Invitrogen</td>
			<td >Q33216</td>
			<td >Section 25</td>
		</tr>
		<tr >
			<td >QIAquick Gel Extraction Kit</td>
			<td >Qiagen</td>
			<td >28704</td>
			<td >Section 25</td>
		</tr>
		<tr >
			<td >Optical Clear Qubit tubes&#160;</td>
			<td >Invitrogen</td>
			<td >Q32856</td>
			<td >Section 26</td>
		</tr>
		<tr >
			<td >Qubit dsDNA High Sensitivity Assay kit</td>
			<td >Invitrogen</td>
			<td >Q32851</td>
			<td >Section 26</td>
		</tr>
	</tbody>
</table>

the chip-exo method: identifying protein-dna interactions with near base pair precision

크로 마틴 면역 침강 (칩)은 후성 유전학 및 특정 단백질 DNA 상호 작용을 분리 유전자 조절의 분야에서 필수적인 도구입니다. 높은 처리량 시퀀싱 (칩 서열)에 결합 된 칩은 일반적으로 염색질 상호 작용 단백질의 게놈 위치를 결정하기 위해 사용된다. 그러나, 칩 서열은 수백 염기쌍 비교적 낮은 매핑 해상도 및 높은 백그라운드 신호에 의해 방해된다. 칩 엑소 방법은 실질적으로 해상도 및 노이즈 모두 개선 한 칩 서열의 정제 된 버전이다. 칩 엑소 방법론의 키 차이는 효과적으로 왼쪽 풋 프린트 라이브러리 준비 워크 플로의 람다 엑소 뉴 클레아 제 소화의 설립 및 단백질 DNA의 가교 사이트의 오른쪽 5 &#39;DNA 테두리입니다. 칩 엑소 라이브러리는 다음 높은 처리량 시퀀싱을 실시한다. 얻어진 데이터는 기능적 조직 O에 고유 초 고해상도 통찰력을 제공하기 위해 활용 될 수있다게놈 바. 여기, 우리는 우리가 최적화 및 포유류 시스템과 차세대 시퀀싱 합성에 의한 플랫폼 간소화 한 칩 - 엑소 방법을 설명합니다.

다음 그림은 여기에 제시된 칩 - 엑소 프로토콜 대표 결과를 보여줍니다. 몇 효소 단계를 기존의 칩-SEQ 방법과는 달리, 칩 - 엑소는 열한 순차적으로 의존 효소 반응 (그림 1)가 필요합니다. 따라서, 치료는 각 반응 성분이 각각의 반응 마스터 믹스에 추가되도록 각 단계에서 수행되어야한다. 우리는 생성 된 표를 인쇄하는 자동 반응 마스터 믹스 계산을 수행하?...

Watch this Scientific Journal Video about The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision at JoVE.com

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision

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 ...

The overall goal of this functional genomic technology is to identify the precise genomic locations that a given protein occupies in living cells. This method can help answer key questions in the fields of gene regulation and epigenetics such as how the transcription machinery assembles and operates in the context of chromatin. The main advantage of this technique is it reveals the exact genomic location for protein DNA interactions at near-base pre-resolution with exceptionally low background.Generally, individuals new to this method will struggle because the protocol is complex and requires attention to detail throughout. To sonicate nuclear lysates, begin by placing resin adapters in 15 milliliter polystyrene tubes containing nuclear extracts. Sonicate the lysates in an ice cold water bath at medium power for 30 seconds on and 30 seconds off for a total of two 15-minute sessions.To check sonication results, transfer two 10 microliter samples from each tube into 1.5 milliliter tubes. Then reverse crosslinks by combining the first 10 microliter sample with 10 microliters of TE-RNase A and 0.2 microliters of proteinase K.Incubate the tube at 37 degrees Celsius for 30 minutes. Then add four microliters of 6X xylene DNA dye.To the second 10 microliter sample, preserve crosslinks by adding two microliters of 6X xylene DNA dye. Run both samples on a 1.5%agarose gel at 140 volts for approximately 30 to 45 minutes until the bromophenol dye in the latter is three-quarters of the way down the gel. If sonication was successful, transfer sonicated lysates to two milliliter tubes containing 150 microliters of 10%Triton X-100 and vortex to mix.Pellet the insoluble chromatin and debris by spinning the sonicated lysates at 20, 000 times g at four degrees Celsius for 10 minutes. Then transfer the supernatants to new two milliliter tubes. Store the samples at negative 80 degrees Celsius indefinitely.For each chromatin immunoprecipitation or ChIP reaction, combine 1.5 milliliters of sonicated extracts with antibody bead conjugates in 1.5 milliliter tubes. Incubate the tubes on a mini tube rotator at a speed of nine at four degrees Celsius overnight. Check after a few minutes to ensure samples are not leaking and are mixing properly.The following day, very briefly spin down the tubes to collect the liquid in the caps and place the tubes on a magnetic rack for one minute. Then aspirate the extract. Add 0.75 milliliters of RIPA buffer to each tube.Then remove the tubes from the magnetic rack and invert them several times to mix. After a brief spin, place the tubes back on the magnet and aspirate the supernatant. Following the last wash, add 0.75 milliliters of 10 millimolar Tris HCL to each tube.Remove the tubes from the magnetic rack and invert the samples several times to mix. After a brief spin, place the tubes back on the rack and aspirate the supernatant. After carrying out the polishing and detailing reactions according to the text protocol, fill out the ligation master mix calculations.Then make the ligation mix in a 1.5 milliliter tube on ice and pipette to mix the solution. With the sample tubes on the magnetic rack, add 48 microliters of P7 ligation master mix and two microliters of a different adapter index to each of the sample resins. Incubate the samples in a ThermoMixer at three times g in 25 degrees Celsius for two hours.After washing the samples and performing the Phy 29 nick repair and kinase reactions according to the text protocol, fill out the lambda exonuclease master mix calculations and make the lambda exonuclease mix in a 1.5 milliliter tube on ice. Add 50 microliters of lambda exonuclease mix to each sample resin while still on the magnetic rack. Then incubate the samples in a ThermoMixer at three times g in 37 degrees Celsius for 30 minutes.After aspirating the last wash as described in the text protocol, carry out RecJf nuclease reaction by filling out the RecJf master mix calculations and making the RecJf mix on ice. Add 50 microliters of the RecJf mix to each sample resin on the magnetic rack. Then incubate the samples in a ThermoMixer at three times g in 37 degrees Celsius for 30 minutes.After aspirating the last wash, prepare the ChIP elution buffer and proteinase K master mix and add 200 microliters of the solution to each sample resin. Incubate the samples in a ThermoMixer at three times g in 65 degrees Celsius overnight with a heated lid to prevent condensation. The following day, briefly spin the samples and place the tubes on the magnetic rack for one minute.Then transfer 200 microliters of supernatant to a new 1.5 milliliter tube containing 200 microliters of TE buffer. Following DNA extraction, P7 primary extension and ATL reactions, fill out the P5 adapter ligation table master mix calculations and make the ligation master mix on ice. Add 20 microliters of the ligation mix to each sample and pipette to mix.Then incubate the samples in a thermal cycler at 25 degrees Celsius for two hours. Carry out DNA purification and quantification according to the text protocol. As shown here, it is worth spending time to optimize sonication conditions to obtain high-quality chromatin extracts that yield DNA fragments between 100 to 500 base pairs.And electrophoretic mobility shift assay is used to verify that the sonicated extracts contain intact protein DNA crosslinks, evident as a super shift of DNA fragments. To assess background signal, a mock ChIP is routinely performed without antibody in the reaction. A high-quality ChIP exo-library will yield very little background in the mock sample compared to a ChIP-seq library preparation.Shown here is a bioanalyzer assessment of a ChIP-exo library. Analysis accurately ensures the library size distribution and detects contaminating adapter dimers that run at 125 base pairs. This panel illustrates the smooth distribution of strand-separated ChIP-exo tag five prime Ns for polymerase II, TFIIB and TBP at the human RPS12 gene in proliferating K562 cells.Shown in this figure are average ChIP-exo patterns relative to the transcriptional start sites of protein coding genes. The major peak locations for TBP, TFIIB and polymerase II in the composite plot reflect the genomic organization of human transcription initiation complexes. Once mastered, this technique can be done in four days if performed properly.After its development, this technique paved the way for researchers in the field of gene regulation to explore molecular details of subnuclear structure in assymetry in yeast. Following this procedure, other methods like RNase seek expression profiling can be performed to address questions such as whether a given factor influences the expression of a nearby gene. While attempting this procedure, it's important to remember to carefully fill out master mix tables for all enzymatic steps.Don't forget, working with carcinogens and other caustic agents can be extremely hazardous and precautions such as wearing personal protective equipment should always be taken while performing this procedure. After this watching video, you should have a good understanding of how to prepare a high-quality ChIP-exo library.

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