iCLIP - 개인 염기 해상도와 단백질 RNA 상호 작용의 Transcriptome 전체 매핑

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Summary

성적표에서 RNA 결합 단백질의 공간적 배열이 포스트 transcriptional 규칙의 주요 결정자이다. 따라서, 우리는 RNA 결합 단백질의 바인딩 사이트의 정확한 게놈 차원의 매핑을 허용 개별 - 염기 해상도 자외선 crosslinking 및 immunoprecipitation (iCLIP)를 개발하였습니다.

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Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., Turner, D. J., Luscombe, N. M., Ule, J. iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. J. Vis. Exp. (50), e2638, doi:10.3791/2638 (2011).

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Abstract

성적표에서 RNA 결합 단백질의 독특한 구성과 공간적 배열 (RBPs)는 포스트 transcriptional 규칙 1의 다양한 측면을 안내입니다. 따라서, 분자 수준에서 명시 규정을 이해 향한 필수적인 단계는 RBPs 2의 바인딩 사이트에 위치 정보를 얻을 수 있습니다.

단백질 RNA 상호 작용은 생화 학적 방법을 사용하여 공부 할 수 있지만, 이러한 방식은 원시 세포 문맥에 바인딩 RNA를 해결하지 않습니다. 자신의 세포 환경에서 단백질 RNA의 단지를 공부하는 초기 시도는 차동 디스플레이 또는 microarray 분석 (RIP 칩) 3-5와 결합 친화력 정화 또는 immunoprecipitation을 고용. 이러한 접근은 간접적 또는 비 생리 상호 작용 6 파악하는 경향이 있었다. 클립 (UV 교차 연결 및 immunoprecipitation) 7,8 도입되면서 특이성과 위치 해상도를 향상시키기 위해, 전략 언급. 클립 UV 가교 denaturing polyacrylamide 젤 전기 영동을 포함한 엄격한 정화 체계와 단백질과 RNA 분자의 결합. 높은 처리량 시퀀싱 기술와 함께, 클립 게놈 전체 규모의 단백질 RNA 상호 작용 (조회수 - 클립이나 CLIP - seq로 함) 9,10을 연구하는 강력한 도구로 입증되었습니다. 최근 PAR - 클립 11,12을 교차 연결을위한 photoreactive ribonucleoside의 analogs를 사용 도입되었습니다.

얻은 데이터의 높은 특이성에도 불구하고, 클립 실험은 종종 제한된 순서 복잡 cDNA 라이브러리를 생성합니다. 이것은 공동 정화 RNA의 제한 금액과 도서관 준비에 필요한 두 가지 비효율적인 RNA의 결합 반응에 일부 때문입니다. 또한, 뇌관 확장 assays 많은 cDNAs의 가교 뉴클레오 티드 13 성급하게 잘릴 것을 지적했다. 이러한 잘린 cDNAs는 표준 클립 라이브러리 준비 프로토콜 동안 손실됩니다. 우리는 최근에보다 효율적인 분자내 cDNA circularization (그림 1) 14과 비효율적인 분자간 RNA의 결합 단계 중 하나를 교체하여 잘린 cDNAs을 캡처 iCLIP을 (개인 - 염기 해상도 클립), 개발했습니다. 중요한 것은 잘린 cDNAs을 시퀀싱하는 뉴클레오 티드 해상도에서 상호 링크 사이트의 위치에 통찰력을 제공합니다. 우리는 성공적으로 게놈 전체의 규모에서 hnRNP C 입자 조직을 연구하고 splicing 규정 14의 역할을 평가할 수 iCLIP 적용.

Protocol

1. UV 교차 연결 조직 문화의 세포

  1. 미디어를 제거하고 10cm 접시 (충분한 세 실험)에 성장 세포 6 ML 얼음처럼 차가운 PBS를 추가합니다.
  2. 얼음 뚜껑과 장소를 제거합니다. 150 254 NM에서 MJ / cm 2 번 비추다.
  3. 휴대 리프터와 스크레이핑하여 세포를 수확.
  4. 세 microtubes 각각 2 ML 세포 현탁액을 전송합니다. 4 10 초에 대한 최고 속도로 회전은 ° C 펠릿 세포에 다음 뜨는 제거합니다.
  5. 스냅 - 냉동 -80에서 드라이 아이스와 저장소에 셀 알약을 ° C까지 사용.

2. 비드 준비

  1. 100 μl의 단백질 새로운 microtube로 실험을 당 Dynabeads (Dynal, 100.02) (마우스 또는 염소 항체에 대한 단백질 G의 Dynabeads를 사용). 추가
  2. . 용해 완충액 (; 100 MM NaCl, 1퍼센트 NP - 40, 0.1 % SDS, 0.5 %의 나트륨 deoxycholate, 1백분의 1 프로 테아제 억제제 칵테일 III, Calbiochem 50 MM 트리스 - HCL, pH를 7.4)로 2 배 구슬 와시
  3. 20-10 μg 항체 100 μl 용해 버퍼에 구슬을 Resuspend.
  4. 30-60 분 실온에서 튜브를 회전합니다.
  5. 900 μl 용해 버퍼 배 세척하고 4.1 단계로 진행 준비가 될 때까지 마지막으로 씻어 둡니다.

3. 세포 용해 및 일부 RNA의 소화

  1. 1 ML의 용해 버퍼와 1.5 ML의 microtubes로 전송에서 세포 펠렛을 Resuspend.
  2. RNase I (앰비온, AM2295)의 500분의 1 희석을 준비합니다. 10 μl RNase I 희석뿐만 아니라 세포 lysate 2 μl 터보 DNase 추가 (500분의 1 RNase I의 dilutions [낮은 RNase] 도서관 준비에 사용되며 50분의 1 dilutions [높은 RNase] 항체 특이성에 대한 제어하는​​ 데 필요한) .
  3. 37 정확히 3 분 ° C, 1100 rpm으로 흔들어에 대한 샘플을 품어. 즉시 얼음으로 전송할 수 있습니다.
  4. 4 스핀 ° C 및 lysate를 지우 20 분 2만2천그램. 조심스럽게 (펠렛과 함께 약 50 μl lysate를 떠나) 표면에 뜨는를 수집합니다.

4. Immunoprecipitation

  1. 비즈 (단계 2.5에서)에서 씻어 버퍼를 제거 후 세포 lysate를 (단계 3.4에서) 추가합니다.
  2. 4 2 H에 대한 샘플을 회전 ° C.
  3. 뜨는을 취소하고 900 μl 높은 소금 버퍼 (50 MM 트리스 - HCL, pH를 7.4, 1 M NaCl, 1 밀리미터 EDTA (에틸렌 다이아 민 테트라 초산), 1% NP - 40, 0.1 % SDS, 0.5 %의 나트륨 deoxycholate)로 2 배 비즈 씻으십시오.
  4. . 900 μl 세척 버퍼 (; 10 MM MgCl 2; 0.2 % 트윈 - 20 20 MM 트리스 - HCL, pH를 7.4)로 2 배 씻으십시오

5. RNA 3'ends의 Dephosphorylation

  1. 뜨는을 취소하고 (15 μl 물, 4 μl 배 PNK의 산도 6.5 버퍼 [350mMTris - HCL, pH를 6.5; 50mMMgCl이 25mMdithiothreitol]; 0.5 μl PNK 효소, 0.5 μl RNasin [Promega]) 20 μl PNK 믹스의 구슬을 resuspend.
  2. 37 20 분 품어 ° C.
  3. 500 μl 세척 버퍼를 추가하고 높은 소금 버퍼 1X 씻는다.
  4. 세척 버퍼와 2X 씻으십시오.

6. 3 '끝을 RNA에 결합 링커

  1. 조심스럽게 뜨는을 제거하고 (9 μl 물을 20 μl 내고 믹스의 구슬을 resuspend, 4 μl 4X 내고 버퍼 [200 mMTris - HCL, 40m의 MM gCl 2; 40 MM의 dithiothreitol], 1 μl RNA ligase [코]; 0.5 μl RNasin [Promega]; 1.5 μl 사전 adenylated 링커 L3 [20 μm의], 4 μl PEG400 [81170, 시그마]).
  2. 16 밤새 품어 ° C.
  3. 500 μl 세척 버퍼를 추가하고 다음 1 ML 높은 소금 버퍼 2X 씻으십시오.
  4. 두 번째 세척 1 ML 1 ML 세척 버퍼와 2X하고 떠나 씻으십시오.

7. RNA 5 '끝 라벨

  1. 뜨는를 제거하고 뜨거운 PNK 믹스 8 μl의 구슬을 resuspend (0.4 μl PNK [코]; 0.8 μl 32 P - γ - ATP, 0.8 μl 10X PNK 버퍼 [코]; 6 μl 물).
  2. 37 5 분 품어 ° C.
  3. 핫 PNK 믹스를 제거하고 20 μl의 1X Nupage 로딩 버퍼 (Invitrogen)에있는 구슬을 resuspend.
  4. ° C 10 분 70시 thermomixer에 품어.
  5. 즉시 (8 단계 참조) 빈 구슬을 촉진하고 겔에 뜨는를 로드할 자석에 놓으십시오.

8. SDS - PAGE와 막 전송

  1. 4~12%의 NuPAGE 제조 업체의 지시에 따라 BIS - 트리스 젤 (Invitrogen)에 샘플을로드합니다. 버퍼 (Invitrogen)를 실행 1X 맙스 0.5 리터를 사용합니다. 또한 사전에 묻은 단백질의 크기 표시 (예 : 페이지의 통치자 또한, Fermentas, SM1811) 5 μl를로드합니다.
  2. 180 50 분 겔을 실행 V.
  3. 겔 전면을 제거하고 고체 폐기물 (무료 방사성 ATP를 포함)로 폐기.
  4. 젤의 제조 업체의 지침 (Invitrogen, 30 V에서 환승 1 H)에 따라 Novex 서부 유럽 표준시 전송 장치를 사용하여 nitrocellulose 막에 단백질 RNA의 단지를 전송합니다.
  5. 전송 후, PBS 버퍼에 막을 린스, 다음 사란 포장에 포장하여 -80 ° C (장소 나중에 T를 정렬하기 위해 막 옆에 형광 스티커를에서 후지 필름에 노출그는 영화와 막이;) 30 분, 1 시간 및 밤 동안에 대한 노출을 수행합니다.

9. RNA 분리

  1. 마스크로 단계 8.5에서 autoradiograph를 사용하여 낮은 RNase 실험에서 단백질 RNA의 단지를 분리. 여러 개의 작은 조각으로 막 조각을 잘라 1.5 ML의 microtube에 그들을 놓으십시오.
  2. 막 조각을 10 μl proteinase K (로체, 03,115,828,001) 200 μl PK 버퍼를 (10 MM EDTA (에틸렌 다이아 민 테트라 초산),, 50 MM 100 MM NaCl 트리스 - HCL의 산도 7.4) 추가합니다. 37 20 분 1,100 rpm으로 흔들어 품어 ° C.
  3. PKurea 버퍼 (; 50 MM NaCl, 10 MM EDTA (에틸렌 다이아 민 테트라 초산), 100 MM 트리스 - HCL pH는 7.4 7 M 우레아) 200 μl를 추가하고 37에서 20 분 품어 ° C.
  4. 솔루션을 수집하고 2 ML 단계 잠금 젤 중공업 튜브 (713-2536, VWR)에 클로로포름 (앰비온, 9722) / 페놀 RNA 400 μl와 함께 추가합니다.
  5. 1,100 RPM에 떨고, 30 5 분 ° C에 대해 품어. 실온에서 13,000 rpm으로 5 분 회전으로 ​​단계를 구분한다.
  6. (피펫로 젤을 만지지 않도록주의) 새로운 튜브로 수성 레이어를 전송합니다. 0.5 μl glycoblue (앰비온, 9510) 40 μl 3 M의 아세트산 나트륨의 산도 5.5와 혼합을 추가합니다. 다음 1 ML 100 % 에탄올을 추가, -20 ° C. 밤 동안 다시 믹스 및 촉진

10. 역방향 전사

  1. 15,000 RPM 4 20 분 스핀 ° C. 뜨는을 제거하고 0.5 ML 80 % 에탄올로 펠렛을 씻으십시오.
  2. . 7.25 μl RNA / 프라이머 믹스 (; 0.5 μl Rclip 프라이머 [0.5pmol/μl]; 0.5 μl dNTP 혼합 [10mM] 6.25 μl 물)의 펠릿을 Resuspend 각각의 실험이나 복제를위한 개별 바코드 시퀀스 (14 참조) 포함된 다른 Rclip의 프라이머를 사용합니다.
  3. 70 5 분 품어 ° C 25 ° C.으로 냉각하기 전에
  4. 2.75 μl RT 혼합 (; 0.5 μl 0.1M DTT, 0.25 μl 윗첨자 III 반전 transcriptase [Invitrogen] 2 μl RT 배 버퍼)를 추가합니다.
  5. 25 5 분 품어 ° C, 42시 20 분 ° C, 50 ° C 40 분 80시 5 분 ° C 4 냉각하기 전에 ° C.
  6. 90 μl TE 버퍼, 0.5 μl glycoblue 10 μl 아세트산 나트륨의 산도 5.5와 혼합을 추가합니다. 그렇다면 250 μl 100 % 에탄올을 추가, -20 ° C. 밤 동안 다시 믹스와 촉진

11. cDNA의 젤 정화

  1. 스핀 다운 및 (10.1 참조) 샘플을 씻어 후, 물 6 μl의 알약을 resuspend.
  2. 6 μl 2X TBE - 요소 로딩 버퍼 (Invitrogen)를 추가합니다. 열 샘플 80 ° C 직접적으로로드하기 전에 3 분.
  3. 프리 캐스트 6% TBE - 요소 젤 (Invitrogen)에 샘플을로드하고 같은 제조 업체에서 설명한 180 V에서 40 분 동안 실행. 또한 이후의 절단 (아래 참조)에 대한 낮은 분자량 마커를로드합니다.
  4. 120-200 NT (높이), 85-120 NT (중간)와 70-85 NT (낮은) 세 밴드 컷. (그림 3 참조) 절단을 안내 theupper 염료 및 플라스틱 겔 지원 부호를 사용하십시오. 참고 클립 순서의 52 NT 용 Rclip 프라이머와 함께 L3 순서 계정입니다.
  5. 400 μl TE를 추가하고 1 ML의 주사기의 플런저를 사용하여 작은 조각으로 겔 슬라이스를 부술. 37 2 H 위해 1,100 rpm으로 흔들어 품어 ° C.
  6. 플레이스 코스타 SpinX 칼럼 (코닝 설립, 8161)에이 1cm 유리 사전 필터 (왓먼, 1823010). 그 결과 해당 컬럼에 대한 샘플의 액체 부분을 전송합니다. 1.5 ML 튜브에 13,000 RPM에서 1 분 스핀.
  7. 0.5 μl glycoblue 40 μl 아세트산 나트륨의 산도 5.5를 추가 후 샘플을 섞는다. 1 ML 100 % 에탄올을 추가, -20 ° C. 밤 동안 다시 믹스 및 촉진

12. cDNA의 5'end에 뇌관을 내고

  1. 그리고 부화 스핀 다운과 (10.1 참조) 샘플을 씻어 후 8 μl 내고 믹스 (; 0.8 μl 10X CircLigase 버퍼 II, 0.4 μl 50 MM MnCl 2;, 0.3 μl Circligase II [Epicentre] 6.5 μl 물)의 알약을 resuspend 60 ° C. 1 H에 대한
  2. 30 μl oligo 소둔 혼합 추가 (26 μl 물, 3 μl FastDigest 버퍼 ​​[Fermentas], 1 μl cut_oligo [10 μm의]). 95에서 1 분 품어 ° C. 그렇다면 1 매 20 초 온도를 감소 ° C 25 ° C까지 도달하고 있습니다.
  3. 37 2 μl BamHI (고속 Fermentas) 30 분 품어 ° C. 추가
  4. 50 μl TE 및 0.5 μl glycoblue 및 혼합을 추가합니다. 10 μl 아세트산 나트륨의 산도 5.5 믹스를 추가한 다음 250 μl 100 % 에탄올을 추가합니다. -20 ° C. 밤 반복 및 석출물 믹스

13. PCR 증폭

  1. 스핀 다운 및 (10.1 참조) 샘플을 씻어 후, 19 μl 물에 펠렛을 resuspend.
  2. (, 1 μl 입문서 믹스 P5/P3 solexa, 10 μm의 각, 20 μl Accuprime Supermix 한 효소 [Invitrogen] 19 μl cDNA)를 PCR 믹스를 준비합니다.
  3. 다음 PCR 프로그램을 실행 25-35 사이클, 68 ° 3 분위한 C, 4 [30 초에 대한 94 ° C 15 초, 65 ° C를위한 30 초, 68 ° C] 94 ° C 2 분을 ° C 영원히.
  4. 프리 캐스트 6% TBE 젤에 배 TBE 로딩 버퍼와 부하 2 μl (Invit 8 μl PCR 제품 믹스로겐). Sybrgreen I (Invitrogen)로 겔 스테인과 젤 영상기와 함께 분석합니다.
  5. Rclip의 primers의 바코드는 높은 처리량 시퀀싱을 위해 제출하기 전에 다른 멀티 플렉스 샘플 수 있습니다. 시퀀싱을위한 라이브러리 15 μl를 제출하고 나머지를 저장합니다.

14. 링커 및 프라이머 시퀀스

사전 adenylated 3 '링커 DNA :

[우리는 20μM의 aliquots을 다음 IDT의 DNA 어댑터를 주문합니다.]

DNA

15. 대표 결과 :

전에 iCLIP 라이브러리의 순서로 실험의 성공은 두 단계에서 모니터할 수 있습니다 : 멤브레인 전송 후 단백질 RNA 복합 (단계 8.5) 및 PCR 제품의 겔 이미지 (단계 13.4)의 autoradiograph합니다. 낮은 RNase 샘플 autoradiograph에서 확산 방사능은 단백질 (그림 2, 샘플 4)의 분자량 위에 볼 수 있습니다. 높은 RNase 샘플의 경우,이 방사능은 가까운 단백질의 분자량 (그림 2, 샘플 3) 초점을 맞추고 있습니다. 어떤 항체가 immunoprecipitation에 사용하지 않은 경우, 어떤 신호는 (그림 2 샘플 1과 2) 감지되지 않을 것입니다. immunoprecipitation의 특이성에 대한 더 중요한 컨트롤 UV 방사선이나 관심 14 단백질을 표현하지 사용 세포를 생략하거나.

PCR 제품 (13.4 단계)의 겔 이미지는 단계 11.4 (그림 4 차선 4-6)에 정화 cDNA 분율 (높은, 중간 또는 낮은)에 해당하는 크기 범위를 표시합니다. PCR primers P3Solexa과 P5Solexa은 cDNA의 크기에 추가 76 NT를 소개합니다. 어떤 항체가 immunoprecipitation 동안 사용하지 않으면, 해당하는 PCR 제품은 (그림 4 차선 1-3) 감지되지 않을 것입니다. 프리머 이합체 제품은 약 140 NT에서 나타날 수 있습니다.

높은 처리량 시퀀싱 및 후속 bioinformatic 분석의 대표적인 결과를 얻으려면 14을 참조하십시오.

그림 1
그림 1. iCLIP 프로토콜의 도식 표현. 단백질 RNA의 단지는 자외선 조사 (1 단계)를 사용하여 생체내에 covalently을 상호 연결되어 있습니다. 관심의 단백질은 바운드 RNA (단계 2-5)와 함께 정화됩니다. 최종 radioactively (6 단계와 7 단계)로 표시됩니다 반대로 전사의 순서를 특정 마중물 수 있도록, RNA 어댑터 '는 5 반면, RNA의 끝'가 3 출혈도 잡았입니다. 교차 연결된 단백질 RNA의 단지는 SDS - PAGE와 멤브레인 전송 (8 단계)를 사용하여 무료로 RNA의 정화입니다. RNA는 proteinase K는 염기 상호 링크 (9 단계)에 남아있는 polypeptide를 떠나있는 단백질을 소화하여 멤브레인에서 발견한 것입니다. 역방향 전사 (RT)는 남아있는 polypeptide로 잘라 두 cleavable 어댑터 지역 및 바코드 시퀀스 (10 단계)을 소개합니다. 크기 선택 circularization 전에 무료 RT의 뇌관을 제거합니다. 다음 선형화은 PCR의 증폭에 적합 템플릿 (11-15 단계)를 생성합니다. 마지막으로, 높은 처리량 시퀀싱은 바코드 시퀀스 즉시 (단계 16) cDNA의 마지막 염기 뒤에있는 읽기를 생성합니다. 교차 연결된 뉴클레오 티드의 상류이 뉴클레오 티드 찾아 한 위치 때문에, 바인딩 사이트는 고해상도로 유추하실 수 있습니다.

그림 2
그림 2. 교차 연결된 hnRNP C - RNA의 단지의 Autoradiograph은 denaturing 겔 전기 영동과 막 전송을 사용합니다. hnRNP C - RNA의 단지가 hnRNP C에 대한 항체를 사용하여 세포 추출물에서 단백질 정화되었습니다 (α hnRNP C, 샘플 3, 4). RNA가 부분적으로 낮은 (+) 또는 높은를 사용하여 소화했다 (+ +) RNase의 농도. 단백질의 크기 (40 KDA)에서 이상 이동 단지는 (예제 4) 볼 수 있습니다. RNase의 높은 농도가 (예제 3) 사용했을 때 변화가 덜 발음됩니다. 어떤 항체가 (예제 1과 2) immunoprecipitation에서 사용하지 때 방사성 신호가 사라집니다.

그림 3
그림 3. 도식 6% TBE - 요소 젤 (Invitrogen)는 iCLIP cDNA 제품의 절단 안내합니다. 겔은 겔에 cDNAs 및 염료 (파란색 조명과 어두운)의 재현성 마이 그 레이션 패턴 최고 180 V에서 40 분 동안 실행됩니다. (빨간색 선) 고 (H), 중간 (M), 낮은 (L) cDNA 분수를 잘라 면도날을 사용합니다. 플라스틱 겔 카세트에 하늘색 염료의 중간에 즉시 마르크 이상 절단하여 시작하십시오. 매체 낮은 분수를 분할하고 하늘색 염료 위 1cm에 대한 높은 분수를 좀 잘라. 다른 차선을 (이 예제 1-4)를 별도로 주머니와 염색으로 보이는 수직 상처를 사용하십시오. 마커 레인 (m)는 스테인드하고 제어하는​​ 데 몇 군데 있습니다절단 후 크기. 조각 크기는 오른쪽에 표시됩니다.

그림 4
그림 4. 겔 전기 영동을 사용하여 PCR - 증폭된 cDNA 라이브러리 iCLIP 분석. RNA는 반대로 베꼈는데 및 크기 정화가 denaturing 겔 전기 영동 (그림 2)를 사용했던 멤브레인 (그림 1)에서 복구. 세 크기 분수 cDNA (높은 [H] : 120-200 NT, 매체는 [M] : 85-120 NT와 낮은 [L] : 70-85 NT) 복구되었습니다, circularized, 다시 선형 및 PCR - 증폭. 다양한 크기의 배포판의 PCR 제품은 입력 분수의 다양한 크기의 결과로 볼 수 있습니다. PCR 프라이머의 cDNA 76 NT를 도입 이래, 크기는 중간 및 낮은 크기의 분수에 대한 146-161 NT 높은, 161-196 NT 용 196-276 NT 사이의 범위해야합니다. 어떤 항체가 immunoprecipitation (1-3 차선)에 대한 사용되지 때 PCR 제품은 결석합니다.

Discussion

iCLIP 프로토콜 효소 반응과 정화 ​​단계의 다양한 범위를 포함하고 있기 때문에, 그것은 실험이 실패 문제를 식별하는 데 항상 쉬운 일이 아닙니다. 확인 RNA 상호 링크 사이트의 특이성에 대한 제어하기 위해서는 하나 이상의 부정적인 컨트롤이 전체 실험 이후 전산 분석을 통해 유지되어야합니다. 이러한 컨트롤은 노 항체 샘플, 비 교차 연결된 셀 또는 녹아웃 세포 또는 조직에서 immunoprecipitation 수 있습니다. 이상적으로, 이러한 제어 실험은 단백질의 RNA 단지를 정화하지 말아야하고, 따라서 SDS - PAGE 젤에 아무런 신호, 및 PCR 증폭 후 노 감지 제품을주지해야합니다. 이러한 컨트롤 라이브러리의 높은 처리량 시퀀싱는 거의 고유의 시퀀스를 반환해야합니다. 결과 시퀀스는 여전히 작은 양이 분해 전지에서 정화되는 것과 동일한 단백질의 상호 링크 사이트에 해당하는 이후 최저 세포는 시퀀싱 제어로 권장하지 않습니다.

주의는 또한 이전의 실험에서 PCR 제품과 오염을 피하기 위해 이동해야합니다. 이 문제를 최소화하는 가장 좋은 방법은 공간 사전 및 사후 PCR 단계를 구분하는 것입니다. 이상적으로, PCR 제품 및 모든 후속 단계의 분석은 별도의 방에서 수행하여야한다. 또한, 실험실의 각 회원은 버퍼와 다른 시약의 자신의 세트를 사용해야합니다. 이러한 방법으로 오염 물질의 출처는 쉽게 확인할 수 있습니다.

Disclosures

관심 없음 충돌 선언하지 않습니다.

Acknowledgements

저자는 토론과 실험 도움 Ule, 루스콤 및 Zupan 연구소의 모든 구성원을 감사드립니다. 우리는 높은 처리량 시퀀싱을위한 제임스 해드와 닉 매튜 주셔서 감사합니다. 우리는 iCLIP 방법은 주, 여기 로버트 다넬의 연구실에서 커크 젠슨과 JU에 의해 개발된 원래의 클립 프로토콜로 여러 단계를 설명 것을 지적하고 싶다. 이 작품은 유럽 연구위원회 부여 JU와 JK에 대한 장기적인 인간 프론티어 과학 프로그램 교제로 206726 - CLIP에 의해 지원되었다

Materials

Name Company Catalog Number Comments
For gel electrophoresis and membrane transfer we recommend t he use of XCell SureLock® Mini-Cell and XCell IIâ Blot Module Kit CE Mark (Invitrogen, EI0002), which is compatible with the use of the different precast minigels that are specified throughout the protocol. The brand and order number of all materials used is mentioned during the protocol. The list of enzymes used in the protocol is shown in the table below.
Protein A Dynabeads Invitrogen 10001D use protein G for mouse or goat antibody
RNase I Ambion AM2295 activity can change from batch to batch
T4 RNA ligase I New England Biolabs M0204S
PNK New England Biolabs M0201S
proteinase K Roche Group 03115828001
Superscript III reverse transcriptase Invitrogen 18080044
Circligase II Epicentre Biotechnologies CL9021K
FastDigest® BamHI Fermentas FD0054
AccuPrime™ SuperMix I Invitrogen 12342010 this PCR mix gives the best results in our hands

DOWNLOAD MATERIALS LIST

References

  1. Keene, J. D. RNA regulons: coordination of post-transcriptional events. Nat Rev Genet. 8, 533-543 (2007).
  2. Wang, Z., Burge, C. B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA. 14, 802-813 (2008).
  3. Trifillis, P., Day, N., Kiledjian, M. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins. RNA. 5, 1071-1082 (1999).
  4. Brooks, S. A., Rigby, W. F. Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique. Nucleic Acids Res. 28, E49-E49 (2000).
  5. Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci. 97, 14085-14090 (2000).
  6. Mili, S., Steitz, J. A. Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA. 10, 1692-1694 (2004).
  7. Ule, J. CLIP identifies Nova-regulated RNA networks in the brain. Science. 302, 1212-1215 (2003).
  8. Ule, J., Jensen, K., Mele, A., Darnell, R. B. CLIP: A method for identifying protein-RNA interaction sites in living cells. Methods. 37, 376-386 (2005).
  9. Licatalosi, D. D. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature. 456, 464-469 (2008).
  10. Yeo, G. W. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol. 16, 130-137 (2009).
  11. Urlaub, H., Hartmuth, K., Lührmann, R. A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles. Methods. 26, 170-181 (2002).
  12. König, J. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol. 17, 909-915 (2010).

Erratum

Formal Correction: Erratum: iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Posted by JoVE Editors on 07/14/2011. Citeable Link.

A correction was made to iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. There was an error in part 2 of step 3. One of the characters had the incorrect symbol and was corrected to:

"...as well as 2 μl Turbo DNase..."

instead of:

"...as well as 2 ml Turbo DNase..."

Comments

71 Comments

  1. Hi,

    First I would like to say this latest method is really neat. I also like Julian's comment at the end of the video when he said with a big smirk, "You have to perform each of the 64 steps with 100% accuracy". :D That is epic.

    On a more serious note, I am just wondering if anyone can suggest what sort of primer I should use if I want to start by cloning my insert into TOPO vector instead of doing nextGen sequencing. Any help is appreciated.

    Paul

    Reply
    Posted by: Anonymous
    June 9, 2011 - 3:47 AM
  2. Hi Paul, thanks for your fun comment! TOPO cloning dŒsn²17;t require any specific primer, so you could use the one described in the protocol. Unless you wish to do something specific, such as concatemerization of sequences before inserting them into vector. Feel free to post more questions! Jernej

    Reply
    Posted by: Anonymous
    June 11, 2011 - 4:19 PM
  3. For more iCLIP questions and answers, use the following Googledoc: http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    June 13, 2011 - 11:28 AM
  4. Hi Jernej,
    Is it possible to use a 3' linker with a phosphorylated 5' end instead of a pre-adenylated 5' end and adding some ATP during the 3' linker ligation step? Thanks. Paul

    Reply
    Posted by: Anonymous
    June 13, 2011 - 10:13 PM
  5. Yes, just follow the protocol as described in Konig et al, NSMB ²010 (PMID ²0601959). More on Googledoc.

    Reply
    Posted by: Anonymous
    June 14, 2011 - 3:48 AM
  6. Hi Jernej,

    Sorry to keep bombarding you with questions. In the supplementary section of your NSMB ²010 paper, shrimp alkaline phosphatase was used to desphosphorylate 3' ends. My understanding is that SAP can only desphosphorylate 5' ends. I am wondering if you had dephosphorylated 3' ends step using PNK before using SAP to dephosphorylate 5' ends.

    Quote from Konig et al, NSMB ²010: "For dephosphorylation of 3²4²; ends, Dynabeads were resuspended in ² µl 10&#²15; Shrimp alkaline phosphatase buffer (Promega), 17.5 µl H²O and 0.1 µl Shrimp alkaline
    phosphatase (Promega) and incubated at 37°C for 10 min with intermittent shaking (10 sec at 700 rpm followed by ²0 sec pause)."

    Thank you again for your help.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 4:04 AM
  7. We did use SAP in the NSMB protocol - it dŒsn't work as well as PNK on the 3' ends. We couldn't use PNK at the time, because PNK carryover into ligation reaction would create problems in the presence of ATP. In jove protocol, ligation reaction lacks ATP, therefore we can use PNK to dephosphorylate the 3' ends.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 6:30 PM
  8. Hi Jernej,

    On 3.² it says add ²ml Turbo DNAse into the 1.5 ml tube. I am wondering if that amount is correct.

    Reply
    Posted by: Anonymous
    July 9, 2011 - 11:45 PM
  9. Hello Paul,
    you are right, it should be two micro liters. Sorry for that, I will try to have it changed,
    Julian

    Reply
    Posted by: Anonymous
    July 10, 2011 - 6:56 AM
  10. Hi Jernej, great protocol! Just a precision, the L3 oligo is a pre-adenylated DNA or RNA oligo? Not clear as the original Clip and iClip uses RNA...

    Thanks a bunch,

    Marco

    Reply
    Posted by: Anonymous
    September 12, 2011 - 3:58 PM
  11. Hi Marco. It's a DNA oligo. Best, Jernej

    Reply
    Posted by: Anonymous
    September 12, 2011 - 4:02 PM
  12. Hi Jernej,

    I am wondering if the 3²P-ATP batch that you normally use in your lab for step 6.1 always has close to a 100% reported radioactivity. What is the lowest percentage of remaining 3²P that you can usually still get away with? I can still get some decent signal when using 3²P-ATP that has ~50-60% remaining radioactivity but my bands on the films are not as intense as the one that I see in your publications. I am trying to work out the best schedule for ordering some 3²P-ATP and starting my experiments. Thanks again.

    Paul

    Reply
    Posted by: Anonymous
    September 14, 2011 - 11:32 PM
  13. We don't use ATP if it's more than two weeks old, thus we have >50% radioactivity. But signal intensity also depends on the efficiency of crosslinking and IP,and amount of protein expression in the cells.

    Reply
    Posted by: Anonymous
    September 15, 2011 - 4:23 AM
  14. Hi Jernej,
    Thank you for the protocol. What results if I reduce the cell samples to 100-1000 (not 10*6-7 cells) ? Thanks for your reply.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 12:24 AM
  15. That would be challenging. If you have an abundant protein that cross-links well to RNA, then it might be possible. So try running the radioactive protein-RNA complex on the gel - if you good signal after overnight exposure, then it's doable.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 4:52 AM
  16. Hi, Jernej !
    Thank you for the reply. I have another questions: How stable if the RNA-RNA and RNA-Protein photocrosslinking? How to degrade these proteins or remove the photocrosslinking? Thank you a lots.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 6:36 AM
  17. Hi Jernej,

    I again have some more questions. Do you still expose the nitrocellulose membrane at -80C when using phosphoimager instead of a film? I'm also wondering what exposure time your lab uses when using a phosphorimager screen.

    Secondly, I am wondering how many libraries containing different barcodes you can run together in a single flow cells.

    Thank you again Jernej. This protocol has been extremely useful.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:49 PM
  18. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  19. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  20. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  21. Hi Jernej,

    In regards to one of the FAQs from Google docs.

    - When analysing PCR products, I see a band corresponding to the size of primer dimers, especially in the sample that was cut low from cDNA gel.

    Yes, it is common to see this band in the sample that was cut low from cDNA gel, and sometimes also in other samples. This is due to contamination from short cDNAs that only contain the sequence of RT primer. If this primer dimer is the dominant product on gel, we advise against sequencing the corresponding sample.

    I seem to be getting this short cDNA contamination all the time. Do you have any advice on how I could try to minimise the contamination? Have you ever isolated fragments of correct-size cDNA from a TBE-urea gel and sent only the isolated fragment for sequencing when you have short cDNA contaminations? Do you think that will work? I think that the concentration of L3 linker that I had used might have been too much. Thank you.

    Reply
    Posted by: Anonymous
    November 22, 2011 - 7:45 PM
  22. There are several possible reasons for this. Maybe one aspect of the protocol is not working, and therefore you are not producing any specific cDNA. If you have no cDNA input, then with enough cycles, you can amplify the primer-primer from any part of the gel. If you are using mammalian cells, try to get the protocol working first with hnRNP C or TIA with Santa cruz antibodies that we used in recent publications. Otherwise, using too much L3 can be a problem.

    Reply
    Posted by: Anonymous
    November 23, 2011 - 4:35 AM
  23. Very useful protocol. I have two questions.

    1. For dephosphorylation of RNA 3'ends, pH 6.5 PNK buffer is used, rather than the pH 7.6 buffer, provided by NEB. Have you compared these two conditions internally?
    ². In the protocol, the final PCR product is not isolated and quantitated before submitting for the sequencing. Are there any potential problems of doing these two steps? Can I isolate the PCR product and re-PCR using the same primers to get more product (for Illumina Hiseq)? Thank you.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 3:40 PM
  24. You can find more related answers in Googledoc http://goo.gl/4tSci, but short answers are also below:

    1. We haven²17;t compared conditions, but increased phophatase activity of PNK at lower pH has been reported in literature, you can read more in the Pubmed ID 1184²1²0.

    ². The PCR product needs to be quantified. We use both qPCR and bioanalyser. Normally, the products of the first PCR should look clean on the gel, otherwise it is a sign of a library that is of low complexity, and is unlikely to generate informative data. Therefore we advise against re-PCR, but it can be done as the last resource.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 4:56 PM
  25. Hi Jernej,

    I noticed you use +/- 10 multiplexed libraries; I was wondering if you knew how many are necessary for a successful run (i.e. to provide sufficient distribution for cluster identification)?

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:20 PM
  26. The way the primers are designed here, no multiplexing is necessary, because the first three nucleotides in the primer sequence are random (part of randomer = NNN).

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:28 PM
  27. I appreciate your experiment. I have some qeustions.

    In this protocol, what dŒs barcode do high-throughout squencing?

    I don't understand function of barcode



    Reply
    Posted by: seung kuk P.
    May 23, 2012 - 6:45 AM
  28. Hi,
    this might be a really naive question but I'm wondering at the UV cross linking step, when you say you irradiate once, dŒ's this mean 1 min?

    Thank you!
    Zsofi

    Reply
    Posted by: Zsofia I.
    June 18, 2012 - 1:19 PM
  29. Hello, Thank you for this helpful technique, I just have a question. My experiments protocols are: 1. UV-crosslink RNA-protein; ². Isolate the RNA-protein complex by immunopricitation; 3. Isolate the binding RNA. 3²P-labeling the binding RNA. 4. Analysis the RNA by microarray.
    Because I do not need to sequence the RNA, and I only want to isolate the binding RNA for microarray analysis after UV-crosslink RNA-protein, so I wonder whether I need to do the step 5-7 in your protocols or I could skip from step 4 to step 8 in your protocol?

    Thanks very much, I look forward to your kind reply!

    Sean

    Reply
    Posted by: xiaoyun w.
    July 22, 2012 - 9:09 PM
  30. It is unlikely you will have enough cDNA for microarray hybridisation without some kind of amplification. You can try using steps 4-8, but you could also amplify in other ways.

    Reply
    Posted by: Anonymous
    July 23, 2012 - 6:03 AM
  31. Hi Jernej,

    Is there any published article on how to analyse iCLIP's high-throughput sequencing data? I have just got my sequencing results back following steps in your protocol. I want to make sure I check with you before digging into the data. Thank you.

    Reply
    Posted by: Anonymous
    August 1, 2012 - 10:15 PM
  32. The article is not yet published, but is in preparation by Tomaz Curk ( http://www.fri.uni-lj.si/en/tomaz-curk/), who made a public server: http://icount.biolab.si/. You can contact Tomaz at tomaz.curk@fri.uni-lj.si for more information.

    Reply
    Posted by: Anonymous
    August 2, 2012 - 5:47 AM
  33. Hi,
    it is so powerful technique! But I cannot IP any protein follow protocol. Is there any difference in affinity between different antibodies and their antigen? Could you give me some advice? Maybe we could decrease concentration of SDS or sodium deoxycholate?
    Thanks, I look forward to your kind reply!
    Min

    Reply
    Posted by: Min S.
    August 5, 2012 - 11:04 PM
  34. Hi Min, you can find advice on IP googledoc http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 6, 2012 - 3:35 AM
  35. Hi Jernej,

    With the barcoding system, I am just wondering if the three random nucleotides are there for indexing purpose during Illumina sequencing run but it's not necessary for splitting the different libraries later on. For RC1, the sequencing results will be something like NNNGGTTNN.... During analysis, do you usually trim the 3-bp from the 5'-end of the results and split the different replicates after the trimming step? I have just realised this was slightly different to the barcoding system used in your NSMB paper. -paul

    Reply
    Posted by: Anonymous
    August 6, 2012 - 9:41 PM
  36. Hi Paul! You can find the answer under the topic of "Use of random barcode in data analysis" in http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 7, 2012 - 7:58 AM
  37. Hi Jernej,

    I started optimising CLIP couple of months ago and I'm at the stage that I'm convinced that I can efficiently cross link RNA to my protein (checked it by specific qRT PCR). I'm lucky because I don't need to fiddle with the IP since I've optimised before and works fine. But just to double check, after IP and western blotting a smear and a lower amount of original kDa protein is a good sing for cross linking yes?
    So my problems started at the RNase A step, I don't see any changes in size/appearance on WB after treatment... I'm convinced that my protein creates a massive complex (couple of 100 kDa) and it is because my target RNA is 10 kb to start with and there are at least 3 proteins binding to it. I'm working with a RNA virus, that's the explanation for it. I think the reason I don't see any change in kDA is because the complex dŒsn't even enter the gel to start up with. Although I used the given buffer which should break any membrane apart but the proteins are still there possibly protecting the RNA. Did you ever come across similar problems and would you have any suggestions? Also, I understand that the RNase trimming is necessary for the efficient RT step but is it a problem if the RNA is too long? What is too long? DŒs this depend on the RT enzyme used I recon or is this also important for the sequencing?

    I would greatly appreciate yur help because I'm stuck...

    Thank you,
    Zsofi

    Reply
    Posted by: Zsofia I.
    October 10, 2012 - 7:38 AM
  38. Hi Zsofi,

    For partial RNAse digestion we use RNase I (step 3). We use two different concentrations: a lower one that makes fragments with a mean between 50-100 bp and a higher concentration that fragments RNA to around 10bp. The lower one is used for preparing libraries, the higher one is used for analytical reasons.

    The RNAse step is important to (1) allow the protein RNA complex enter the Gel (²) to narrow down the crosslink site to a fragment with a size compatible with high throughput sequencing (maximum around 300 bp). So you definitely need to optimize this step for your experiments.

    If the complex you are studying is not covalently linked it should fall apart during the denaturing Gel run. Only a small fraction of your complex will have all the proteins of your complex crosslinked to the RNA at the same time since crosslinking is a very inefficient step. Therefore with the higher RNAse concentration you should be able to see a radioactive signal at the size of the protein you are studying.

    I hope that helps, best regards,
    Julian

    Reply
    Posted by: Julian K.
    October 11, 2012 - 9:49 AM
  39. Hi Julian,

    I have had some trouble with the RNase step when nuclease-ing the total lysate... In my troubleshooting efforts I read that RNase I is inhibited by 0.1% SDS, which is the concentration used in your lysis buffer. It dŒsn't seem that you guys have any problem though...do you think this is due to using an excess of RNase I or what? Just curiously confused. Thanks,

    sam

    Reply
    Posted by: Sam F.
    February 5, 2013 - 6:00 PM
  40. Hi Sam,

    in our experience the inhibition of RNase I by SDS is not an issue. You just optimize the concentration of RNase I to obtain the desired fragmentation. If you have problems doing that with your buffer conditions, you could also do the RNase digestion on the beads instead of in the lysate.

    Best,
    Julian

    Reply
    Posted by: Julian K.
    February 6, 2013 - 6:19 AM
  41. Thanks for the quick reply Julian. Your recommendation to do the "on bead" digestion is exactly what I have done and it seems to be working fine. Cheers

    Posted by: Sam F.
    February 6, 2013 - 10:12 AM
  42. Hi,

    I was wondering how many minutes have you irradiated the cells in case of HNRNP C?

    Reply
    Posted by: Niaz M.
    November 26, 2012 - 6:29 PM
  43. Hi Niaz,
    we are normally not measuring time of irradiation but the Energy per square centimeter:
    Step 1.²: ... Irradiate once with 150 mJ/cm² at ²54 nm.
    In our Stratalinker this takes 50s. However time of irradiation is not very informative here since it changes with the age or quality of the lamps, etc.
    Cheers, Julian

    Reply
    Posted by: Julian K.
    November 27, 2012 - 6:20 AM
  44. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  45. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  46. Hi Lisa,

    it is correct that the ends of P3 and P5 primers are the same. This is because of Illumina's primer design for their high throughput sequencing platform. When you look at the 3' end of the Rclip primers (after the Bamhi cleavage site) you can see that they are actually complementary to the 3'end of the L3 adapter.

    Cheers,
    Julian

    Reply
    Posted by: Julian K.
    December 18, 2012 - 10:05 AM
  47. I got it !!!
    Thank you :)

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 1:11 PM
  48. Hi
    Thanks for the protocol. I have one question that has been bothering me, though. Both the RNA ligase and PNK buffers will expose the antibody column to relatively high dithiothreitol (DTT) concentrations (10 mM and 5 mM respectively). Why dŒsn't this destroy the column by reducing the disulphide bonds holding the heavy and light antibody chains together? Have you ever tried to improve the immunoprecipitation step by attempting to minimize the DTT concentration as much as possible or is this not an issue. Any assistance would be greatly appreciated. Thanks - Greg

    Reply
    Posted by: Greg C.
    February 3, 2013 - 2:13 PM
  49. Hi Greg, we haven't seen an effect of the DTT in the buffers on the IP efficiency, it seems that the concentration is not high enough to reduce the IgG - however, it is worth testing this the first time you do IP, since it is plausible that this will vary dependent on the source of your buffers (company used for PNK and ligase), or antibodies.

    Reply
    Posted by: Anonymous
    February 6, 2013 - 2:45 AM
  50. Hi Jernej,
    Thanks for the reply. The antibody I am using is definitely sensitive to the level of DTT found in the PNK buffer and I need to limit the over-all exposure of the column to DTT as much as possible. As a result, rather than using PNK as the 3' phosphatase, I would like to use an alkaline phosphatase. I noticed that in your ²010 NSMB paper you are using Shrimp Alkaline Phosphatase and in your ²009 Methods paper you use FAST AP. Did you find that the Shrimp phosphatase is significantly better ?

    Thanks again - Greg

    Reply
    Posted by: Greg C.
    February 8, 2013 - 3:52 PM
  51. Hi Greg, we don't have any evidence to suggest that one is better than the other for the on-bead reaction. At the time we were using SAP in the lab generally since it can be heat-inactivated, so therefore we also used it for on-bead (even though here you can't heat-inactivate it on beads). So you can go ahead with either one.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:29 AM
  52. I should also add that even though we didn't compare FAST AP and SAP, we did compare SAP with PNK, and we had a lot better results with PNK. It seems that SAP is not efficient as a 3' phosphatase. So it may be better for you to determine the minimal DTT amount in the buffer that is compatible with your antibody, and then continue using it with PNK and ligase. If you use fresh DTT, 1mM is likely to be sufficient both for PNK and RNA ligase.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:39 AM
  53. Thanks, I really appreciate the advice.
    -Greg

    Posted by: Greg C.
    February 9, 2013 - 9:03 AM
  54. Hi, thanks for the awesome video. I have two questions related to the reagents:
    1. What concentration is the PEG400? (it only says 4 ul in the protocol).
    ². Under "Reverse transcription", step 6, what is the pH of the TE buffer you use? Is it pH 8?
    Thank you very much for your help. -QT

    Reply
    Posted by: Qiumin T.
    February 7, 2013 - 1:22 PM
  55. Hi QT,
    (1) we are using PEG400 from Sigma (²0²398). It is a viscous liquid.
    (²) Yes, the it is pH 8
    Cheers, Julian

    Reply
    Posted by: Julian K.
    February 7, 2013 - 4:56 PM
  56. Thank you so much Julian. I have another question. Could you recommend a protocol for doing iCLIP with mouse brain tissue? Do you know whether the tissue prep steps from this protocol ( http://ago.rockefeller.edu/Ago_HITS_CLIP_Protocol_June_²009.pdf) will work well for iCLIP as well?

    Reply
    Posted by: Anonymous
    February 13, 2013 - 5:52 PM
  57. This protocol should be fine. We also recently published a bookchapter about the iCLIP protocol which contains information on tissue samples and lots of other useful info and background:
    http://onlinelibrary.wiley.com/doi/10.100²/97835²764458².ch10/summary

    Reply
    Posted by: Julian K.
    February 14, 2013 - 5:19 AM
  58. The pre-publication version of the book chapter is available here: http://www².mrc-lmb.cam.ac.uk/groups/jule/publications/Konig_wiley.pdf.

    Reply
    Posted by: Anonymous
    February 19, 2013 - 1:54 PM
  59. I enjoyed reading about your updated iCLIP protocol in the book Tag-based Next Generation Sequencing from Wiley. I would be grateful for more details about the amount and activity of the 3²-P that you use to radioalabel the RNA. In both the book chapter and the JoVE article I see only volumes, not activities.

    In the figure for step 9, the radiolabel on the 5' end of the RNA is missing, but shouldn't it still be there? At what point in the protocol can we be reasonably sure that we are dealing with unlabeled material?

    Also, have you ever explored non-radioactive approaches to labeling, or is the sensitivity of these methods too low for the purposes of this protocol?

    Thanks!

    John

    Reply
    Posted by: John S.
    June 17, 2013 - 9:23 AM
  60. Dear John,

    with the current protocol most of the radioactivity is gone after the gel purification of the cDNA. You can increase this effect by treating the samples with RNAse after the reverse transcription (The radioactive RNA fragments then running much faster then the cDNAs in the gel). We are currently working on a protocol where we fragment the RNA by alkaline hydrolysis, which will be available soon (We want to avoid using too much RNAse at our desks).

    In addition you should always measure your samples with a Geiger counter. If your final PCRs are still hot, then you should decrease the fraction of beads that go into the labeling reaction.

    Best wishes,
    Julian

    Reply
    Posted by: Julian K.
    June 19, 2013 - 11:52 AM
  61. Hi,
    I have a question which relies on your experience with the data generated with CLIP:
    because of the UV irradiation the protein crosslinks to the RNA which even after proteinase K treatment presents an obstruction to the reverse transcriptase which therefore either skips or adds random nucleotide(s). So my question is that how long the deletions/insertions can be? Is it only one nucleotide or can also be 20?

    Thank you very much!!!

    Reply
    Posted by: Zsofia I.
    August 8, 2013 - 12:06 PM
  62. We see that >80% of cDNAs truncate at the crosslink site, and the mutations are quite rare in the remaining sequences. All we know about crosslink-induced mutations has been published here: http://www.ncbi.nlm.nih.gov/pubmed/22863408.

    Reply
    Posted by: Anonymous
    August 8, 2013 - 12:15 PM
  63. Thanks for your protocol. I have a question about the IgG background signal in the p32 labeled Western Blot. I used mouse IgG1 isotype as a control. I did not crosslink the IgG to the beads, so IgG1 stays around 50 and 25 KDa region. I do observe some radioactivity band around 50 kDa. Do you notice this in your experiments as well? Since it is close to my protein region, can you give me some suggestions to avoid this?

    Sincerely,
    Mei

    Reply
    Posted by: Xuemei Z.
    August 9, 2013 - 1:42 PM
  64. We don't get a signal in control IP. Most likely this is an RBP that non-specifically binds under your conditions. It is important to wash with high-salt buffer, and rotate the tubes for ±5min during these washes. Also, diluting the lysate before IP may help. Standard IP optimisations, basically.

    Reply
    Posted by: Anonymous
    August 9, 2013 - 2:24 PM
  65. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:29 AM
  66. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:57 AM
  67. Hi,
    I have 2 more questions. In this protocol you did not remove 5' phosphate of the RNA, can you still label the 5' side with P32 by PNK later? Another question, is it possible to just p32 label the RNA, cut the band, extract, degrade the protein and add 3' linker for RT later?

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:06 PM
  68. Normal PNK has phosphatase activity, so it can replace the 5' phosphate. The original CLIP protocol from Ule et al, Science 2003 added 3' linker after RNA extraction, but as explained in Ule et al, Methods 2005., the efficiency and purity of the protocol increases if linker is ligated on beads.

    Reply
    Posted by: Anonymous
    August 21, 2013 - 2:15 PM
  69. Thanks for this amazing protocol and your rapid and very helpful exchange here in this site.

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:26 PM
  70. Hello, thank you for this wonderful protocol.
    I have a question:
    -I get positive Radioactive signal at the right size of positive CTRL used in this protocol in the NOT UV samples, it looks exactly as I was using high RNAse condition. why?
    I am phosphorylating the protein? is it possible?
    thank you

    Reply
    Posted by: jessica c.
    February 27, 2016 - 8:45 PM
  71. Hi Jessica, you are right, if you see signal in the non-UV control, this means that the protein is getting phosphorylated in some other way. If it has a kinase domain it may even phosphorylate itself. Or maybe some kinase is getting co-purified? You could check for this by omitting PNK from the phosphorylation reaction.

    Reply
    Posted by: Jernej U.
    March 16, 2016 - 11:07 AM

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