작품은 필라델피아의 아동 병원에서 연구원 개발 포상에 의해 지원됩니다.

1. 상호 링크 염색질 <ol><li> 100x20mm 세포 배양 접시에 세포를 성장. 세포의 양이 1 세포의 종류에 따라 접시 당 1,000 만 셀에 이르기까지 다양하실 수 있습니다. 약 2 백만 세포는 하나의 면역 침전 충분합니다. </li><li> 가끔 흔들 실온에서 10 분 동안 1 %의 포름 알데히드의 가교 세포. </li><li> 상온에서 5 분 동안 125 mM의 글리신과 부화의 최종 농도를 추가하여 상호 연결을 끄다. </li><li> 1X 인산염과 세포를 씻으은 가만히 따르다 PBS, 두 번 식염수 (PBS)를 버퍼, 그리고 PBS의 0.2 ML를 추가합니다. </li><li> microcentrifuge 관에 플라스틱 셀 스크레이퍼 수확 세포. </li><li> 4에서 5 분간 2,000 rpm으로 세포를 스핀 다운 ° C. </li><li> 뜨는을 대기음. 전체 셀에 대한 SDS 용해 버퍼에있는 세포를 resuspend (1 % SDS, 10MM EDTA의 50mm 트리스 - 염산 산도 8.1) 해물이나 핵 추출 펠렛으로 보관하십시오. </li><li> 세포 ° C 또는 하나의 immediat를 진행할 수 -80 저장할 수 있습니다초음파와 엘리. </li></ol> 2. (전체 세포 파쇄물을 위해 3.5 단계로 진행) 핵을 준비 <ol><li> 1X 단백질 분해 효소 억제제 각 실험로 (5 개 mm 파이프 산도 8.0, 85 MM의 KCl을, 0.5 % NP-40) 세포 용해 버퍼를 보충합니다. </li><li> 약 10 배 세포 용해 버퍼 펠릿 볼륨 해동 세포 펠렛을 resuspend을. </li><li> 유 봉 다운스 - 균질화 10 배는 10 분 동안 얼음에 품어. </li><li> 4에서 5 분 ° C에 대한 4,000 rpm에서 샘플을 원심 분리기, 상층 액을 버리고, 핵 펠렛을 저장합니다. </li></ol> 3. 초음파 * <ol><li> SDS의 용해 버퍼 및 단백질 분해 효소 억제제로 사용되는 버퍼의 보조 금액을 따뜻하게. </li><li> SDS 용해 버퍼를 Resuspend 핵 펠렛 (1-10000000 세포 당 버퍼의 0.5 ML) </li><li> 10 분 동안 얼음에 품어. </li><li> 의 microcentrifuge 튜브에 샘플 0.5 ML의 aliquots를 추가합니다. </li><li> 초와 45 초 30로 Misonix의 sonicator를 사용하여 젖은 얼음에 초음파 처리오프 2의 진폭 설정에서. 이상적인 조각의 크기 사이클 수를 먼저 다양한 사이클 번호 (예 : 2, 4, 8, 12, 16, 20 이상주기)을 시도하여 확인할 수 있습니다. sonicator의 다른 브랜드는, 그러나, 조건이 달라집니다 사용할 수 있습니다. 주기에 시간의 금액의 번호와 실험 및 해제는 이상적인 조건을 결정하기 위해 수행해야합니다. </li><li> 초음파 결과를 확인하고, 정량화를 수행하기 위해 각 시료 20 μl를 수집합니다. 샘플의 나머지는 -80 ° C.에 저장할 수 있습니다 </li><li> 0.1X TE 버퍼 30 μl를 추가하여 샘플의 20 μl를 희석. </li><li> RNase가 1 μL로 샘플을 치료 37 1 시간 동안 ° C는 2 시간 동안 62 ° C에서 단백질 분해 효소 K와 부화의 1 μl를 추가합니다. </li><li> 2 % 아가로 오스 겔에 시료 20 μl를 실행합니다. </li><li> 다음 Nanodrop 분광 광도계를 사용하여 정량화 QIAquick PCR 정화 키트와 샘플의 남은 양을 정화. </li></ol> * 네이티브 치에 대한P, microccocal 핵산의 소화 전단 DNA를에 선택적으로 사용할 수 있습니다. 4. 차단 아가로 오스 비즈 * <ol><li> 구슬이 이미 차단 된 경우, 5.1 단계로 진행합니다. </li><li> 단백질 또는 단백질 G 아가로 오스를 사용합니다. 5 immunoprecipitations를 들어 IPS (), 50 %의 비드 슬러리 (300 μL 비드 펠렛) 600 μl를 사용 </li><li> 구슬을 씻어, 4 ° C에서 1 분간 800 rpm으로 그들을 회전하고 상층 액을 버린다. 추가 약간 더 2보다 ML 칩 희석 버퍼 (0.01 % SDS, 1.2 mM의 EDTA, 167 mM의 NaCl을 1.1 % 트리톤 X-100, 16.7 MM 트리스 - 염산 산도 8.1) 천천히 반전 튜브 10X로 섞는다. 4 ° C에서 1 분간 800 rpm으로 다시 스핀 다운 및 뜨는을 폐기하십시오. 세척 2 번 이상 반복합니다. </li><li> 솔루션을 차단에 4 ° C에서 하룻밤 회전 구슬을 차단합니다. 차단 솔루션의 조리법은 표 1을 참조하십시오. </li></ol> 5. 사전 맑은 염색질 <ol><li> 해동 얼음에 염색질을 초음파. </li><li> 12,000 RPM의 F에서 스핀 다운또는 4시 10 분 ° C 다음 SDS (흰색 펠렛)를 제거하는 즉시 얼음에 넣어. </li><li> 상층 액을 수집, 펠렛을 폐기하고, 필요한 경우 샘플을 결합합니다. </li><li> 계산 (IP 당 염색질의 1-10 UG)에 따라 실험에 필요한 금액을 꺼냅니다. </li><li> 단백질 분해 효소 억제제와 보완 칩 희석 버퍼에 배 염색질을 희석. </li><li> IP 당 차단 구슬의 100 μl를 추가합니다. </li><li> 1 시간 동안 4 ° C에서 회전합니다. </li></ol> 6. 면역 침전 <ol><li> 1 분간 800 rpm으로 샘플을 스핀 다운과 신선한 튜브에 뜨는을 전송합니다. </li><li> 1 분 다른 깨끗한 튜브로 전송을 위해 800 rpm으로 상등액 스핀 다운. </li><li> -20 ° C.에서 입력 컨트롤 역할을, 상층 액 20 μl를 저장 </li><li> 실험에서 수행 할 IP의 개수로 나누어지는 염색질합니다. </li><li> 각 샘플에 염색질의 1-10 UG 당 항체의 2 UG를 추가합니다. </li><li> 회전 하룻밤 4 ° C를 품어. </li><li> 각 IP 샘플 차단 비즈 100 μl를 추가합니다. </li><li> ° C 회전 4에서 1 시간 동안 배양한다. </li><li> 펠릿 1 분 동안 800 rpm으로 스핀 다운 가능한 한 상층 액만큼을 취소하여 구슬. </li><li> 낮은 소금 면역 복합체 세척 버퍼를 한 번 구슬을 씻으십시오. 각 튜브에 버퍼 1 ML을 추가, 5-8 분 동안 상온에서 회전, 1 분 동안 800 rpm으로 스핀 다운 후 상층 액을 버린다. 높은 소금 면역 복합체 세척 버퍼 LiCl을 면역 복합체 세척 버퍼를 한 번 씻어 반복 회 5 세척 (표 2)의 총 TE 버퍼와 함께. </li></ol> 7. 용출 <ol><li> 전날 해동 입력 샘플 eluants으로 처리 할 수​​ 있습니다. </li><li> 용출 버퍼 신선한 (표 3)을 확인합니다. </li><li> IP를 및 입력 컨트롤 샘플 플러스 1-2 추가 샘플에 필요한 충분한 용출 버퍼의 마스터 믹스를 확인하십시오. </li><li> 각 IP 샘플 100 μL 용출 버퍼를 추가, 15 m에 대하여 상온에서 배양회전합니다. </li><li> 1 분간 800 rpm으로 스핀 다운과 신선한 튜브에 뜨는을 추가합니다. </li><li> 구슬의 각 튜브에 용출 버퍼의 또 다른 100 μl를 추가하고 회전 15 분간 실온에서 알을 품다. </li><li> 부화 후 15 초 동안 소용돌이, 1 분 동안 5,000 rpm으로 스핀 다운, 다음 첫 번째 용출의 상층 액과 상층 액을 결합합니다. (무 상층 액에있는 구슬을 통해이 남아 있는지 확인합니다. 확실하지 않으면 1 분간 5,000 rpm에서 다시 상층 액을 아래로 회전하고 새로운 튜브에 뜨는를 수집합니다. </li><li> 입력 제어 샘플 20 μL로 용출 버퍼 180 μl를 추가합니다. </li></ol> 8. 상호 링크를 역방향 <ol><li> eluants 및 입력 컨트롤의 200 μL에, 5 M NaCl을 8 μl를 추가합니다. </li><li> 65 ° C 하룻밤 물에 목욕 파라 필름 (parafilm)와 부화와 인감 튜브. </li></ol> 9. DNA 정제 <ol><li> 37 1 시간 동안의 RNase의 1 μL 각 샘플을 치료 ° C. </li><li> 4 μ 추가0.5 M EDTA (에틸렌 다이아 민 테트라 초산), 13 μL 1M 트리스 - 염산, 혼합,의 L는 2 시간 동안 45 ° C에서 각각의 샘플과 부화에 단백질 분해 효소 K 1 μl를 추가합니다. </li><li> QIAquick PCR의 정제 키트를 사용하여 샘​​플을 정화. 샘플은 추후에 수행 할 수 있습니다 -20 ° C와 PCR 검사에 저장할 수 있습니다. </li></ol> * 또한, 칩 고급 자석 구슬은 면역 침전 부분에 대한 아가의 장소에서 사용할 수 있습니다. 10. PCR 검사 <ol><li> PCR 검사에 대한 관심의 단백질에 결합하는 것으로 알려져 지역을 위해 프라이머를 사용합니다. 또한, 음성 대조군으로 구속력이 지역의 프라이머를 사용합니다. </li><li> 반응 시약을 혼합합니다. 1:100 (표 3)에서 입력 샘플을 희석. </li><li> 반응을 실행합니다. PCR 프로그램 : </li></ol> 1 단계 : 94 ° C 3 분 2 단계 : 94 ° C 20 초 59 ° C 30 초 72 ° C 30 초 (적어도 30 CYCL에 대해 2 단계를 반복ES) 3 단계 : 72 ° C 2 분 <ol start="4"><li> 1 % 아가 로스 겔에서 샘플을 실행합니다. </li><li> 농축은 실시간 PCR을 정량적으로 측정 할 수 있습니다. </li></ol>

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M., 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=The+8q24+cancer+risk+variant+rs6983267+shows+long-range+interaction+with+MYC+in+colorectal+cancer.">The 8q24 cancer risk variant rs6983267 shows long-range interaction with MYC in colorectal cancer.</a> Nature Genetics. 41, 882-884 (2009).</li><li>Tuupanen, 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=The+common+colorectal+cancer+predisposition+SNP+rs6983267+at+chromosome+8q24+confers+potential+to+enhanced+Wnt+signaling.">The common colorectal cancer predisposition SNP rs6983267 at chromosome 8q24 confers potential to enhanced Wnt signaling.</a> Nature Genetics. 41, 885-890 (2009).</li><li>Zhao, J., Schug, J., Li, M., Kaestner, K. H., Grant, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease-associated+loci+are+significantly+over-represented+among+genes+bound+by+transcription+factor+7-like+2+(TCF7L2)+in+vivo.">Disease-associated loci are significantly over-represented among genes bound by transcription factor 7-like 2 (TCF7L2) in vivo.</a> Diabetologia. 53, 2340-2346 (2010).</li><li>Benjamini, Y., Yekutieli, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+Loci+analysis+using+the+false+discovery+rate.">Quantitative trait Loci analysis using the false discovery rate.</a> Genetics. 171, 783-790 (2005).</li></ol>

제작자들은 더 경쟁 금융 이익이 없다는 점 선언합니다.

아주 최근에 다른 전사 인자 2,3로 입증되었습니다로서, 지금은 칩 SEQ를 사용하여 단백질 DNA 상호 작용 협회의 게놈 전체 프로필을 수행 할 가능합니다. 성공적으로 시퀀싱 결과의 핵심은 높은 품질의 염색질의 immunoprecipitation의 DNA 템플릿의 세대입니다. DNA 템플릿을 생성하고 적절하게 풍성하게 할 ascertained되면, 하나는 후속 시퀀싱 라이브러리 준비에 걸릴 수 있습?...

몇 년 동안, 특히 특정 단백질 게놈 넓은 전사 인자 클래스의 사람들에 의해 바인딩 조절 유전자의 집합을 식별하는 unmet 필요가있다. 오돔 등. 1 체계적으로 인간의 간 및 췌장에서 미리 지정된 전사 규제에 의해 점령 유전자를 식별하는 발기인 마이크로 어레이와 함께 염색질의 immunoprecipitation을 (칩)를 사용. 그 후, 존슨 등 2. 종합적으로 전체 포유류의 게놈에 걸쳐 단백질 DNA 상호 작용을 매핑하기 위해 직접 매우 높은 처리량 DNA 시퀀싱 (칩 SEQ)를 기반으로 대규모 염색질 immunoprecipitation의 분석을 개발했습니다. 테스트 케이스로, 그들은 생체 내에서 인간 게놈 1946 년 위치에 신경 제한적인 소음 계수 (NRSF)의 바인딩을 매핑됩니다. 데이터는 내가 모두를 촉진 바인딩 위치 선명한 해상도 (+ 50 염기쌍)를 표시주제 및 NRSF 결합 모티프의 식별 solation. 이 칩 SEQ 데이터는 높은 민감도와 특이도 및 통계적 신뢰도 (P &lt;10 -4), 새로운 후보 상호 작용을 추론에 중요한 특성을 가지고 있었다. 로버트슨 등. 3도 STAT1 대상에 매핑하기 위해 칩 SEQ를 사용하여 인터페론-γ (IFN-γ) 자극 및 생체 내에서 인간의 헬라 S3 세포를 자극받지. 칩 서열, 15.1 및 12,900,000 고유 매핑 순서 읽고, 0.001의 추정 거짓 발견 속도를 사용하여, 그들은 각각 자극과 자극받지 않은 세포에서 41,582 및 11,004 추정 STAT1 결합 영역을 확인했다. STAT1 인터페론 반응 결합 부위 4-8를 포함하는 것으로 알려진 34 유전자 좌의, 칩 SEQ 24 (71 %)를 발견했다. 칩 SEQ 대상은 알려진 STAT1 바인딩 모티프와 유사한 서열에 충실 하였다. 기존의 두 칩 PCR 데이터와 비교 제안 설정이 칩 SEQ 민감도는 70 %와 92 %, 특이도했다 적어도 95 % 사이였다. 또한, 그것은 칩 SEQ 깊이를 연속으로 증가 낮은 분석의 복잡성과 민감성을 모두 제공한다는 것은 분명했습니다. 등 &quot;차세대&quot;게놈 시퀀싱 기술은 9 이전 기술을 통해 생성 된 비용 효율적으로 할 수 순서의 양에 크기가 증가 1-2 주문을 제공하기 때문에. 칩 SEQ 방법은 따라서 직접 포유류 단백질 DNA 상호 작용 3 효과적인 프로파일에 대한 전체 게놈의 범위를 제공합니다. 2006 년 전사 인자 2 7처럼 (TCF7L2) 제 2 형 당뇨병 유전자 변형의 강한 협회는 10 발견되었다. 다른 연구자들은 이미 독립적으로 자연 11,12에 발표 된 제 2 형 당뇨병의 첫 번째 게놈 넓은 협회 연구에서, 흥미롭게도 다른 인종이 발견을 복제하고있다 </s다른&gt; 과학 13 ~ 15 및 16, 17 위로, 강한 협회는 TCF7L2와 참이었다;이 지금은 18 ~ 20 연대를 제 2 형 당뇨병에서 가장 중요한 유전 발견으로 간주됩니다. 또한, TCF7L2은 암 위험 21,22에 연결되었습니다, 대장 암 등 암의 수의 게놈 넓은 협회 연구에 의해 밝혀 8q24 현장은 극도의 상류에 의한 것으로 표시되었을 때 실제로,이 연결은 더 분명되었다 TCF7L2 바인딩 요소는 MYC 23,24의 전사를 운전. 등이 주요 전사 인자에 의해 조절 하류 유전자를 결정에 큰 관심이있다. 방법의 예로 TCF7L2 경험을 바탕으로, 본 논문은 높은 품질의 칩 DNA 템플릿을 생성하는 방법을 설명합니다. 칩은 높은 레솔을 빌드 순서에서 다음 순서를 위해, 대장 암 세포주 HCT116에서 실시되었다복잡한 특성의 병인에있는 그것의 중요한 역할에 추가 통찰력을 얻을 수있는 노력에 TCF7L2 (25)에 의해 구속 유전자의 ution지도.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAquick PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28104 </td>
 <td></td>
 </tr>
 <tr>
 <td>EZ-ChIP Kit </td>
 <td>Millipore </td>
 <td>17-371 </td>
 <td></td>
 </tr>
 <tr>
 <td>GoTaq Hot Start Polymerase </td>
 <td>Promega</td>
 <td>M5001</td>
 <td></td>
 </tr>
 <tr>
 <td>Misonix Sonicator</td>
 <td>Qsonica </td>
 <td>XL-2000 </td>
 <td></td>
 </tr>
 <tr>
 <td>
 NanoDrop 1000 Spectrophotometer </td>
 <td>
 Thermo-Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Positive control primer sequences (TCF7L2-1) 
 Forward- 5'-TCGCCCTGTCAATAATCTCC-3' 
 Reverse- 5'-GCTCACCTCCTGTATCTTCG-3' 
 Negative control primer sequences (CTRL-1) 
 Forward-5'-ATGTGGTGTGGCTGTGATGGGAAC-3'
 
 Reverse- 5'-CGAGCAATCGGTAAATAGGTCTGG-3'</td>
 </tr>
 </tbody>
</table>

generation of high quality chromatin immunoprecipitation dna template for high-throughput sequencing (chip-seq)

염색질의 immunoprecipitation (칩)을 결합하고 대규모 병렬 염기 서열은 생체 내 전사 인자에 의해 구속 포유류 DNA 서열의 레퍼토리를 식별하기 위해 활용 될 수있는 칩 시퀀싱 (칩 SEQ) 방법은 직접 전체 게놈 범위를 제공합니다. &quot;다음 세대&quot;게놈 시퀀싱 기술은 할 수 순서의 양에 크기가 증가 1-2 순서를 제공하는 비용 효율적 따라서 직접적으로 포유류의 효과적인 프로파일에 대한 전체 게놈의 범위를 제공하는 칩 SEQ 방법을 허용하는 오래된 기술을 통해 생성 단백질 DNA 상호 작용. 성공적인 칩 서열 방식의 경우, 하나는 고품질의 칩 DNA 템플릿 최적의 시퀀싱 결과를 얻기 위해 생성해야합니다. 설명이 가장 강하게 제 2 형 당뇨병, 즉 전사 인자의 전사 인자 2 7처럼 (TCF7L2의 병인에 관여하는 유전자의 단백질 제품과 경험을 기반으로합니다). 이 요소는 각종 암에 연루되어있다. 설명은 병인의 핵심 역할에서 더 통찰력을 제공, TCF7L2 구속 유전자를 확인하는 순서를 통해 고해상도지도를 구축하기 위해 대장 암 세포주 HCT116에서 파생 된 고품질의 칩 DNA 템플릿을 생성하는 방법입니다 복잡한 특성의.

염색질은 초음파되어 RNase의와 단백질 분해 효소로 처리되고 나면, 2 % 아가로 오스 겔에서 실행되는 샘플은 원하는 크기의 DNA 대량으로 얼룩을 제시해야한다. 여러 가지 사이클을 테스트하는 경우 크기가 점차 감소 사이클 수가 증가 (그림 2)로 볼 수 있어야합니다. 프로토콜의 면역 침전 부분을 완료 한 후 농축은 어느 PCR 또는 실시간 PCR 확인할 수 있습니다. 아?...

Watch this Scientific Journal Video about Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq at JoVE.com

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

염색질의 immunoprecipitation과 매우 높은 처리량 시퀀싱 (칩 SEQ)의 조합은 특정 조직이나 세포주에서 단백질 DNA 상호 작용을 파악하고 매핑 할 수 있습니다. 설명은 예를 들어 전사 인자 TCF7L2 경험을 사용하여 다음 순서를위한 고품질 칩 템플릿을 생성하는 방법이다.

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 ...

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Biology

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

24.3K 조회수.  Children's Hospital of Philadelphia Research Institute. 염색질의 immunoprecipitation과 매우 높은 처리량 시퀀싱 (칩 SEQ)의 조합은 특정 조직이나 세포주에서 단백질 DNA 상호 작용을 파악하고 매핑 할 수 있습니다. 설명은 예를 들어 전사 인자 TCF7L2 경험을 사용하여 다음 순서를위한 고품질 칩 템플릿을 생성하는 방법이다.

비디오: Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

높은 처리량 시퀀싱에 의한 마이크로 RNA 정량을위한 작은 RNA 분자의 고효율 내고

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

연구

생물학

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

DNA 메틸화 분석 차세대 시퀀싱 대상

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

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

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

애기 장대 단백질 DNA 상호 작용의 식별을위한 염색질 면역 침전 분석<em&gt; 생체</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-SEQ : 아데닌 - 메틸화 된 DNA 단편의 높은 처리량 시퀀싱에 의해 단백질 DNA 상호 작용의 게놈 넓은 매핑

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

레트로 바이러스 통합 사이트의 증폭, 차세대 시퀀싱 및 게놈 DNA 매핑

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

토마토 털이 뿌리의 생성에 의한 높은 처리량 CRISPR 벡터 건설 및 DNA 수정의 특성

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

높은 처리량 시퀀싱을 활용하여 전체 - 단백질 포화 돌연변이 유발 도서관의 기능 평가를위한 프로토콜

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

현장 별 DNA 분열을 위한 병렬 높은 처리량 단 분자 운동 분석

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

Modified Methods for Loading of High-Throughput DNA Extraction Plates Reduce Potential for Contamination

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, host characteristics, and broader ecosystem functions. With this rapid increase in breadth and depth of sequencing capabilities have come methods to extract, amplify, analyze, and interpret environmental DNA successfully with maximum efficiency. Unfortunately, performing DNA extractions quickly can come at the cost of increasing the risk of contamination among samples. In particular, high-throughput extractions that are based on samples contained in a 96-well plate offer a relatively quick method, compared to single-tube extractions, but also increase opportunities for well-to-well cross-contamination. To minimize the risk of cross-contamination among samples, while retaining the benefits of high-throughput extraction techniques, we developed a new method for loading environmental samples into 96-well plates. We used pierceable PCR sealing films to cover each plate while loading samples and added samples first to PCR tubes before moving them into wells; together, these practices reduce the risk of sample drift and unintended double loading of wells. The method outlined in this paper provides researchers with an approach to maximize available high-throughput extraction techniques while reducing the risk of cross-contamination inherent to 96-well plates. We provide a detailed step by step outline of how to move from sample collection to DNA extraction while minimizing the risk of unwanted cross-contamination.

Current methods for loading 96-well plates for DNA extractions can be prone to contamination. We detail a new method for loading 96-well plates that reduces risk of cross-contamination among wells. Our method will help other researchers capitalize on the efficiency of high-throughput DNA extraction techniques and minimize risk of contamination.

고처리량 DNA 추출 플레이트의 로딩을 위한 수정된 방법은 오염 가능성을 감소시니다.

High-throughput DNA sequencing techniques have contributed substantially to advances in our understanding of relationships among microbial communities, ...