저자는 싱가포르의 * 스타에 의해 지원됩니다. 또한, MJF는 과학 국립 원정대와 리 Kuan 흔히 묘지에 심는 상록수 포스트 박사 원정대의 여성에 대한 * STAR 국가 과학 장학금, 로레알에 의해 지원됩니다. YR은 NIH 인코딩 기금 (R01 HG004456-01와 R01 HG003521-01)에 의해 지원됩니다. 저자는 또한 비디오 편집 및 대한 부인 미셸 테오에 대한 양 Siti 라힘, 장면 촬영을 위해 특정 켈빈 씨 Issey 씨가 장 카이 시앙 씨는 Sherwin Gan 8 픽셀 프로덕션, 싱가포르의 videography 팀을 인정 성우.

<ol>
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관심의 어떠한 충돌 선언 없습니다.

ChIA-PET는 전사 조절에 장기적인 상호 작용을 파악하기 위해 개발된 방법입니다. ChIA-PET 라이브러리의 품질을 결정하는 중요한 요인 중 하나는 칩 재료의 품질입니다. 동영상에 표시된 프로토콜은 세포를 상호 연결 EGS와 포름 알데히드의 사용을 포함합니다. 긴 스페이서 팔을 베어링 두 번째 가교 시약과 함께 포름 알데히드의 사용은 혼자 3,11,15 포름 알데히드에 의해 구속 수없는 단백질의 바인딩에 도움이 될 수 있습니다. 우리는 강력한 구속력 사이트와 원거리 상호 작용 9를 증명하고있다이 방법으로 도서관을 건립했습니다. 그러나, 교차 연결 및 칩 조건 관심의 각 요소에 최적화된되어야하고, 가교 너무 많이는 sonication하여 조각의 어려움을 초래하므로 그렇지 과잉 crosslink 중요하게, 그리고 아마도 가짜 염색질 상호 작용을 초래할 수 . 염색질 상호 작용ChIA-PET으로 식별이 같은 형광에 원위치 하이브리드화 4와 같은 다른 방법에 의해 검증되어야한다. 우리는 염색질 소재의 100 NG의 최소 권장합니다. 우리는 염색질 소재의 50 NG에서 양질의 라이브러리를 구축했지만, 우리는 시작 물질의 다량이 허용하는 나쁜 자들이 미만 16 PCR주기,이를 최소화 amplicons과 각 도서관의 중복과 ChIA-PET 라이브러리의 건설. 이것은 낮은 중복되므로 시퀀싱의 적은 차선과보다 포괄적인 염색질 상호 작용지도를함으로써, 높은 고유의 매핑 태그와도 사용할 데이터의 높은 비율로 상호. 각 튜브에있는 구슬의 최종 포장 부피는 각각 자석과 세파 로스 구슬 50 μl, 100 μl 있어야합니다. 포장 비드 볼륨이 명시된보다 작으면 DNA 베어링 구슬의 손실을 최소화하기 위해 유사하게 사전 허가 빈 자성이나 세파 로스 구슬과 최소 포장 볼륨 가져후속 단계들. 잘라 버렸군 오프 팁 또는 대형 코어 팁은 세파 로스 구슬을 pipetting 동안 사용해야합니다. 다음과 같은 수정은 이전에 다음 ID로 출판 ChIA-PET 프로토콜 다섯 따라 통합되었다. 첫째, 자기 G 비즈는 세차장 중에 샘플의 손실을 최소화하기 위해 사용되었습니다. 또한, 우리는 스스로 출혈도 잡았 반 linkers 또는 / 및 어댑터의 amplicons로 100 BP 138 BP의 대략적인 크기가 아닌 특정 밴드를 발견. 따라서, 우리는 PCR 증폭하는 동안이 아닌 특정 대역을 최소화하기 위해 biotinylated 반 linkers 및 454 GS20 어댑터의 농도를 감소. 근접 내고 볼륨은 이후 정제 단계 중에 샘플의 손실을 최소화하고 또한 시약 비용을 절감 50 ML에서 10 ML로 축소되었다. 우리는 또한 streptavidin 비즈에서 ChIA - 애완 동물의 DNA 최대한의 캡처를 보장하기 위해 구슬로 ChIA-PET DNA를 고정하기 위해 배양 시간이 증가하였습니다. 근접 내고 단계 동안, 키메라 ligations THAt는 필연적 아닌 구체적이고 랜덤 방식으로 생성되는 생체내 염색질의 상호 작용에 진실을 대표하지 않습니다. 따라서, 어떤 ChIA-PET 실험에서 데이터의 품질을 평가하기 위해 chimerism의 비율은 특정 염기 바코드 TAAG 및 ATGT 5 두 개의 다른 반 linkers의 사용으로 추정된다. 높은 처리량 시퀀싱 후 ChIA-PET 시퀀스가 먼저 8을 구분할 수 특정 내고 제품 및 불특정 내고 제품에서 파생된 링커 바코드 조성과 시퀀스에 대해 분석하고 있습니다. 알려진 chimeras의 비율 (즉, heterodimers 순이 linkers)는 우리 내부에 MCF-7 RNA 중합 효소 II의 ChIA-PET 라이브러리 미만 15 % 제시. ChIA-PET 시퀀스가​​ 연속적으로, 즉 두 종류, 자기 내고 애완 동물과 인터 내고 애완 동물로 분류됩니다. 상호 내고 애완 동물이 전에서 파생되는 동안 셀프 내고 애완 동물은 염색질 조각 자체 circularization의 결합에서 얻은됩니다서로 다른 두 가지의 DNA 조각 사이 nter-내고 있습니다. 후자 그러면 동일한 염색체 (intrachromosomal 상호 내고 애완 동물)에 또는 둘 다 태그가 두 개의 다른 염색체 (interchromosomal 상호 내고 애완 동물)에 매핑됩니다 각 태그의 게놈 거리에 따라 세 가지 범주로 하위 나눈 값입니다. 우리는 서로 다른 범주에게 8 해결할 ChIA-PET 도구 소프트웨어 패키지를 개발했습니다. 이것은 라이브러리에있는 DNA 조각을 기준으로합니다. 일반적으로, 작은 칩 조각은 높은 해상도를 제공하고 4킬로바이트에 관한 이러한 RNA 중합 효소 II의 ChIA-PET 라이브러리에 대해 차단됩니다. 또한, 실제 염색질 상호 작용은 상호 작용 클러스터에서 상호 결합 애완 동물의 수를 세어 랜덤 노이즈로부터 구별할 수 있으며, 즉, 높은 PET 개수의 클러스터가 실제 염색질 상호 작용 8이라는 높은 확률을 가지고 있다고합니다 . 우리의 잘못된 반응을 필터링하려면무작위로 우연히 간 내고 애완 동물을 형성할 수 매우 풍부 앵커에서 상승, 통계 분석 프레임 워크는 또한 두 앵커 8 사이의 상호 결합 애완 동물을 무작위로 형성 용도에 대한 상세한 설명이 공식화되었습니다. 결론적으로 ChIA-PET 기술은 세계적인 규모 염색질 상호 작용 네트워크를 매핑 수 있습니다. ChIA-PET의 칩 구현은 도서관 복잡 배경 잡음의 감소 수 있습니다. 또한,이 칩은 특정 전사 인자 5과 관련된 구체적인 염색질 상호 작용 시험있게 염색질 상호 작용에 특이성을 추가합니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 4~20% 구배 TBE 겔 </td><td> Invitrogen </td><td> EC6225BOX </td><td> B, 6.3 단계 </td></tr><tr><td> PEG 5 X T4의 DNA Ligase 버퍼 </td><td> Invitrogen </td><td> 46300018 </td><td> b 단계, 2.2 </td></tr><tr><td> 6% TBE 겔 </td><td> Invitrogen </td><td> EC6263BOX </td><td> B, 6.8 단계 </td></tr><tr><td> 애질런트 DNA 분석 100 </td><td> 애질런트 테크놀로지 </td><td> 5067-1504 </td><td rowspan="3"> b 단계, 7.1 </td></tr><tr><td> 애질런트 고감도의 DNA 분석 </td><td> 애질런트 테크놀로지 </td><td> 5067-4626 </td></tr><tr><td> 애질런트 Bioanalyzer 2100 </td><td> 애질런트 테크놀로지 </td><td> G2940CA </td></tr><tr><td> (스핀-X) 튜브 필터 원심 분리기 </td><td> 코닝 &lt;/ TD&gt; <td> CLS8160 </td><td> 단계 B, 3.7 및 6.9 </td></tr><tr><td> 다크 리더 Transilluminator </td><td> 클레어 화학 연구 </td><td> DR46B </td><td> B, 6.8 단계 </td></tr><tr><td> 디지털 Sonifier 세포 장애 </td><td> 브랜 </td><td> 450D-0101063591 </td><td> , 4.3 단계 </td></tr><tr><td> DynaMag-2 자석 (마그네틱 입자 농축기) </td><td> Invitrogen </td><td> 123-21D </td><td> 단계를 닦고 모든 자석 구슬을 위해,, 7.6.1 단계 B를 단계 </td></tr><tr><td> DynaMag-15 자석 (마그네틱 입자 농축기) </td><td> Invitrogen </td><td> 123.01D </td><td> , 5, 6 단계 </td></tr><tr><td> DynaMag-PCR (마그네틱 입자 농축기) </td><td> Invitrogen </td><td> 49-2025 </td><td> B, 6.5 단계 </td></tr><tr><td> 대장균의 DNA의 중합 효소 I </td><td> 코 </td><td> M0209 </td><td> B, 5.6 단계 </td></tr><tr><td> GlycoBlue </td><td> Ambion </td><td> AM9516 </TD&gt; <td> , 7.6.1 단계 B, 4.3 및 6.5 단계 </td></tr><tr><td> Illumina cBot 클러스터 발전 시스템 </td><td> Illumina </td><td> SY-301-2002 </td><td> 단계 B, 7.4 </td></tr><tr><td> Illumina 게놈 분석기 IIx </td><td> Illumina </td><td> SY-301-1301 </td><td> 단계 B, 7.5 </td></tr><tr><td> Illumina PE primers </td><td> Illumina </td><td> PE-102-1004 </td><td> 단계 B, 5.4 (필요한 경우) </td></tr><tr><td> Intelli-믹서 </td><td> Palico 생명 공학 </td><td> RM-2L </td><td> , 회전 어떤 incubations를 B 단계 </td></tr><tr><td> LightCycler 480 리얼 타임 PCR 시스템 </td><td> 로슈 </td><td> 04 640 268 001 </td><td> , 7.5.3 b 단계, 7.3 단계 </td></tr><tr><td> LightCycler480 DNA SYBR 녹색 나는 MasterMix </td><td> 로슈 </td><td> 03 752 186 001 </td><td> 단계 B, 7.5.3 </td></tr><tr><td> M-280 Streptavidin Dynabeads </td><td> Invitrogen </td><td> 11206D </td><td> B, 5.2 단계 </td></tr> &lt;TR&gt; <td> 마그네틱 (Dynabeads) 단백질 G </td><td> Invitrogen </td><td> 100.03D </td><td> , 5.1.1 및 5.2.1 단계 </td></tr><tr><td> 고밀도 (2ml)을 MaXtract </td><td> Qiagen </td><td> 129,056 </td><td> , 7.6.1 단계 </td></tr><tr><td> 고밀도 (50ml)을 MaXtract </td><td> Qiagen </td><td> 129,073 </td><td> 단계 B, 4.2 </td></tr><tr><td> MmeI </td><td> 코 </td><td> R0637 </td><td> R0637 </td></tr><tr><td> RNase 한 Ribonuclease </td><td> Promega </td><td> M426C </td><td> 단계 B, 4.8 </td></tr><tr><td> RNA 중합 효소 II (8WG16) 단클론 항체 </td><td> Covance </td><td> MMS-126R </td><td> , 5.2.4 단계 </td></tr><tr><td> 오크 릿지의 원심 분리기 튜브 (폴리 프로필렌) </td><td> Nalgene </td><td> 3119-0050 </td><td> , 3.1 단계 </td></tr><tr><td> 오크 릿지의 원심 분리기 튜브 (테플론 FEP) </td><td> Nalgene </td><td> 3114-0050 </td><td> 단계 B, 4.3 </td></tr><tr><td> Phus이온 하이 피델리티 마스터 믹스 </td><td> Finnzymes </td><td> F-531 </td><td> B, 6 단계 </td></tr><tr><td> Picogreen (양의-IT) dsDNA 시약 </td><td> Invitrogen </td><td> P11495 </td><td> , 7.5.2 단계 </td></tr><tr><td> 폴리스티렌 라운드 히프 테스트 튜브 </td><td> BD Biosciences </td><td> 352,057 </td><td> , 4.1 단계 </td></tr><tr><td> 테아제 억제제 칵테일 알약 (완료, EDTA (에틸렌 다이아 민 테트라 초산) - 무료) </td><td> 로슈 </td><td> 11873580001 </td><td> 1.6 이후 단계 </td></tr><tr><td> Proteinase K 솔루션 (20mg/ml) </td><td> Fermentas </td><td> E00491 </td><td> 7.6.1, 4.4 단계 b 단계, 4.1 </td></tr><tr><td> T4의 DNA Ligase </td><td> Fermentas </td><td> EL0013 </td><td> 단계 B, 2.2, 3.9 및 5.4 </td></tr><tr><td> T4 DNA Ligase 버퍼 (코) </td><td> 코 </td><td> B0202S </td><td> b 단계, 3.3 및 3.9 </td></tr><tr><td> T4의 DNA 중합 효소 </td><td> Promega </td><td> M4215 </td><td>b 단계, 1.2 </td></tr><tr><td> T4의 DNA 폴리 뉴클레오 타이드의 키나제 </td><td> 코 </td><td> M0201 </td><td> B, 3.3 단계 </td></tr><tr><td> TruSeq SBS 키트 V5-GA </td><td> Illumina </td><td> FC-104-5001 </td><td> Illumina 게놈 분석기 IIx 시스템 Regents </td></tr><tr><td> TruSeq PE 클러스터 키트 V2-cBot-GA </td><td> Illumina </td><td> PE-300-2001 </td><td> Illumina cBot 클러스터 발전 시스템과 함께 사용 될 </td></tr><tr><td> SYBR 녹색 전 </td><td> Invitrogen </td><td> S-7585 </td><td> b 단계, 6.3 및 6.8 </td></tr><tr><td> XCell SureLock 미니 셀 전기 영동 시스템 </td><td> Invitrogen </td><td> EI0001 </td><td> b 단계, 6.3 및 6.8 </td></tr></tbody></table><table border="1"><tbody><tr><td> 이름 </td><td colspan="2"> 순서 </td><td> 댓글 </td></tr><tr><td rowspan="2"> Biotinylated 반 linkers (200ng/μl) </td><td> 상단 </td><td> 5 &#39;GG CCG CGA / iBiodT / ATC TTA TCC AAC 3&#39; </td><td> 250 nmole 규모의 HPLC 정화 내부 비오틴 DT (9) </td></tr><tr><td> 봇 </td><td> 5 &#39;GTT GGA TAA GAT ATC GC 3&#39; </td><td> 250 nmole 스케일 HPLC 정화 </td></tr><tr><td rowspan="2"> Biotinylated 반 linkers B (200ng/μl) </td><td> 상단 </td><td> 5 &#39;GGC CGC GA / iBiodT / ATA CAT TCC AAC 3&#39; </td><td> 250 nmole 규모의 HPLC 정화 내부 비오틴 DT (9) </td></tr><tr><td> 봇 </td><td> 5 &#39;GTT GGA ATG 두드리는 ATC GC 3&#39; </td><td> 250 nmole 스케일 HPLC 정화 </td></tr><tr><td rowspan="2"> 비 biotinylated 반 linkers (200ng/μl) </td><td> 상단 </td><td> 5 &#39;GGC CGC GAT GGA TCC ATC AAC 3&#39; </td><td> 250 nmole 규모 PCR 학년 </td></tr><tr><td> 봇 </td><td> 5 &#39;GTT GGA TCC GAT ATC GC 3&#39; </td><td> 250 nmole 규모 PCR 학년 </td></tr><tr><td rowspan="2"> GS20 아답터 (200ng/μl) </td><td> 상단 </td><td> 5 &#39;CCA TCT CAT CCC TGC GTG TCC CATCTG TTC CCT CCC TGT CTC AGN N 3 &#39; </td><td> 250 nmole 규모 PCR 학년 </td></tr><tr><td> 봇 </td><td> 5&#39;CTG AGA 배속 GGA GGG AAC AGA TGG GAC ACG 배속 GGA TGA GAT GG 3 &#39; </td><td> 250 nmole 규모 PCR 학년 </td></tr><tr><td rowspan="2"> GS20 아답터 B (200ng/μl) </td><td> 상단 </td><td> 5 &#39;CTG AGA CAC GCA ACA GGG GAT AGG CAA GGC ACA 배속 GGG ATA GG 3&#39; </td><td> 250 nmole 규모 PCR 학년 </td></tr><tr><td> 봇 </td><td> 5 &#39;CCT ATC CCC TGT GTG CCT TGC CTA TCC CCT GTT GCG TGT CTC AGN N 3&#39; </td><td> 250 nmole 규모 PCR 학년 </td></tr><tr><td rowspan="2"> Illumina-NN 어댑터 </td><td> 상단 </td><td> 5 &#39;ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TC 3&#39; </td><td> 250 nmole 규모 HPLC 정화는 단계 B, 5.4 참조 </td></tr><tr><td> 봇 </td><td> 5 &#39;phos - GAT CGG AAG AGC GGT GCA TCA GGA ATG CCG AG 3&#39; </td><td> 250 nmole 규모 HPLC 정제는 5 &#39;최종 단계 B가 5.4 참조 Phosphorylated </td></tr><tr><td> Illumina 1-454 (전진) 프라이머 (10 μm의) </td><td colspan="2"> 5 &#39;AAT GAT ACG GCG ACC ACC 개그 ATC 전술 ACC CTA TCC CCT TGC GTG CTT G 3&#39; </td><td> 250 nmole 규모 PCR 학년 </td></tr><tr><td> Illumina 2-454 (리버스) 프라이머 (10 μm의) </td><td colspan="2"> 5 &#39;CAA GCA GAA GAC GGC ATA CGA GAT CGG TCC ATC TCA TCC CTG C​​GT GTC 3&#39; </td><td> 250 nmole 규모 PCR 학년 </td></tr><tr><td> qPCR의 프라이머 1.1 (10 μm의) </td><td colspan="2"> 5 &#39;3 시요 AAT GAT ACG GCG ACC ACC 개그&#39; </td><td> 10nmole 규모 PCR 학년 </td></tr><tr><td> qPCR의 프라이머 2.1 (10 μm의) </td><td colspan="2"> 5 &#39;CAA GCA GAA GAC GGC ATA CGA 3&#39; </td><td> 10nmole 규모 PCR 학년 </td></tr><tr><td> Illumina 3-454 시퀀싱 프라이머 (100 μm의) </td><td colspan="2"> 5&#39;TGC GTG TCC CAT CTG TTC CCT CCC TGT CTC AG 3 &#39; </td><td> 100nmole 스케일 HPLC 정화 </td></tr><tr><td> Illumina 4-454 시퀀싱 프라이머 (100 μm의) </td><td colspan="2"> 5&#39;GTG CCT TGC CTA TCC CCT GTT GCG TGT CTC AG 3 &#39; </td><td> 100nmole 규모HPLC 정화 </td></tr></tbody></table> 통합의 DNA 기술 (IDT)와 같은 방식으로 linkers 및 어댑터를 준비부터 oligos 및 어댑터의 테이블. 주문 oligos 이전에 10 설명했다. 하프 linkers 및 어댑터는 -20 ° C.에서 몇 달 동안 미리 준비하고 저장될 수 있습니다

chromatin interaction analysis with paired-end tag sequencing (chia-pet) for mapping chromatin interactions and understanding transcription regulation

Genomes은 마이크론 크기의 핵 대 7 내부의 고차원 conformations, 2, 12, 도입, 3 차원 구조로 구성됩니다. 이러한 아키텍처는 무작위 아닌 유전자 발기인 및 규제 요소 13 사이의 상호 작용을 포함. 구체적인 규제 시퀀스에 전사 요소의 바인딩은 전송 규제와 조정 1, 14 네트워크에 대해 제공합니다. 이점 엔드 태그 장면 (ChIA-PET)에 의한 염색질 상호 작용 분석은 이러한 고차원 염색질 구조에게 5,6를 식별할 수 있도록 개발되었습니다. 세포 고정하고 상호 작용하는 loci는 공유 결합의 DNA-단백질 상호 링크에 의해 점령된다. 불특정 노이즈를 최소화하고 복잡성을 줄일뿐만 아니라, 염색질 상호 작용 분석의 특이성을 높이기 위해 위해 염색질 immunoprecipitation (칩)은 근접 내고 전에 관심 염색질 조각을 풍부하게하기 위해 특정 단백질 요인에 대해 사용됩니다. 하프 linkers 관련된 내고 그 후 개별적인 염색질의 단지 내에 함께 곁에의 DNA 조각의 쌍 사이에 공유 결합 링크를 형성하고 있습니다. 하프 linkers의 측면 Mm​​eI 제한 효소 사이트는 MmeI는 소화시 이점 끝 태그 - 링커 - 태그 구조 (애완 동물)의 추출이 가능하다는 특징을 가지고있다. 하프 linkers는 biotinylated하므로 이러한 PET 구조는 streptavidin-자석 구슬을 사용하여 정화하고 있습니다. 정화 애완 동물 차세대 시퀀싱 어댑터와 상호 작용하는 조각의 카탈로그 같은 Illumina 게놈 분석기와 같은 차세대 sequencers 통해 생성된과 출혈도 잡았있다. 매핑 및 생물 정보학 분석은 다음 여덟 사이트 칩 농축 바인딩 및 칩 농축 염색질 상호 작용을 파악하기 위해 수행됩니다. 우리는 칩의 품질 ChIA-PET 라이브러리의 결과에 중요한 역할을 담당로서 특히 ChIA-PET 프로토콜, 칩의 준비의 중요한 측면을 설명하는 동영상을 제작했습니다. 프로토콜은 것처럼오래만이 중요한 단계는 비디오에 표시됩니다.

Watch this Scientific Journal Video about Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation at JoVE.c

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

이점 엔드 태그 장면 (ChIA-PET)에 의한 염색질 상호 작용 분석을위한 방법입니다<em&gt; 드 노보</emtranscriptional 제어의 이해를위한 염색질의 상호 작용&gt; 감지,.

염색질 상호 작용을 매핑 및 전사 규정을 이해하는 데 이점 엔드 태그 장면 (ChIA-PET)과 염색질의 상호 작용 분석

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

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Biology

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

30.5K 조회수.  Agency for Science, Technology and Research, Singapore. 이점 엔드 태그 장면 (ChIA-PET)에 의한 염색질 상호 작용 분석을위한 방법입니다 드 노보 감지,.

비디오: Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

인간 두뇌 조직에 대한 염색질 어세이

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

연구

생물학

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

10 나노 그램 총 RNA의 단일 읽기와 결합하여 최종 mRNA - Seq의 Illumina 도서관

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

사용 염색질 Immunoprecipitation (CHIP) Drosophila 조직

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA 정제에 의한 염색질 절연 (처프)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

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

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

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

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

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

전체 마운트 난자에있는 염색질 수정 및 핵 구조의 정량, 단일 세포 분석을위한 효율적인 방법<em&gt; 애기 장대</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

세대와 인간의 INO80 염색질 리모델링 단지 및 서브 콤플렉스의 정화

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...

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 ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

특정 게놈 지역과 enChIP으로 관련 분자의 식별의 분리

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).