The authors would like to thank members of the Yi laboratory especially Zhaojie Zhang for fruitful discussions regarding linker design and ligation efficiencies, as well as the American Cancer Society for supporting this work through a postdoctoral fellowship (#125209) to J.E.L. Research reported in this publication was also supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR059697 (to R.Y.) and a research grant from the Linda Crnic Institute for Down Syndrome. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

참고 : 전체 과정 동안의 RNase가없는 상태를 유지하는 것이 중요합니다. 3 &#39;링커 1. Adenylation <ol><li> 뉴 클레아 제 무 H 2 O. 100 μM 3 &#39;라이 게이션 반응을 위해 설계된 DNA 올리고 뉴클레오타이드 희석 참고 : 올리고 뉴클레오티드는 5 &#39;인산기, 5에서 무작위 뉴클레오티드&#39;끝, 및 3 &#39;dideoxycytosine이 있어야합니다. 5 adenylation 10 &#39;인산기 효율적인 5 M의 효소의 요구 사항이다.&#39; 포스페이트 직접 변형과 폴리 뉴클레오티드 키나아제와 함께 올리고 뉴클레오티드의 예비 처리에 의해 첨가되거나 주문할 수있다. 5 &#39;말단에서 무작위 뉴클레오티드는 특정 최종 끝이 다른 6 이상 반응에 합류하기위한 RNA 리가 제는 전시 순서 기본 설정을 최소화하는 데 중요합니다. 3 올리고 뉴클레오티드 t의 &#39;말단 dideoxycytosine는 (3)에 차단 부재를 배치&#39;오 후속 결찰 단계 동안 링커 링커 concatamers의 형성을 억제하고 또한 연결 반응 (11) (3 &#39;) (3)의 단부에 형성된 miRNA에 링커 분자의 말단&#39;을 차단하는 역할을한다. 다음 편견은 miR-SEQ 방법을 위해 선택된 올리고 뉴클레오티드 링커 서열은 다음 5&#39;PO 4 NNTGGAATTCTCGGGTGCCAAGG-dideoxyC3 &#39;. </li><li> 200 pmol의 3 &#39;링커 올리고 뉴클레오티드, 4 ㎕의 10 배 5&#39;DNA의 adenylation 완충액 4 ㎕의 1 mM의 ATP, 4 ㎕의 M의 RNA 리가 제, 뉴 클레아 제 무 H 2 O 40 ㎕의 최종 부피로 다음 adenylation 반응을 조립한다. </li><li> 1 시간 동안 65 ° C에서 반응을 품어. 5 분 동안 85 ° C로 가열하여 반응을 종료합니다. </li><li> 잔류 ATP를 제거하기 위해 미래 반응에서 예상치 못한 결찰 않도록 100 % 에탄올 2.5 볼륨과 10 M 암모늄 아세테이트의 1/3 양을 첨가하여 석출 adenylated 링커. <ol><li> 간단히, 알을 혼합cohol, 소금, 20 분 동안 4 ° C에서의 microcentrifuge에서 최고 속도로 회전 동질성에 올리고 솔루션 (~ 15,000 XG). adenylated 올리고 50 ~ 100 μM을 산출하기 위해 핵산 분해 효소가없는 H 2 O의 적절한 볼륨에 70 % 에탄올, 공기 건조 및 재현 탁에서 펠렛을 씻으십시오. </li></ol></li><li> 18 % 폴리 아크릴 아마이드 젤을 실행하여 adenylation을 확인합니다. <ol><li> 다음 함께 혼합하여 겔을 준비 : 6.3 g 우레아, 40 % 아크릴 아미드 6.75 ㎖, 10 배 및 1.5 ml의 TBE. H 2 O 15 ML을 추가합니다. </li><li> 우레아가 완전히 용해 될 때까지 혼합하고 암모늄 퍼 설페이트 (APS) 및 15 μL의 tetramethlyethylenediamine (TEMED) (/ V w) 150 μl의 10 %를 추가한다. </li><li> 겔은 1 배 TBE 실온에서 버퍼를 실행하는 겔 폭에 24 V / cm에서 30 분간 사전 실행을 설정하면. 버퍼를 실행 30 분까지되면 플러시 우물. </li><li> 와 adenylated 올리고 뉴클레오티드 및 배 변성 RNA 로딩 버퍼의 동량 (95 % (V / V) 포름 아미드 5 pmol의 혼합0.02 % SDS, 0.02 % 브로 모 페놀 블루 (V / W), 0.01 % (V / W) (W / V) 크실렌 Cyanol, 1.0 mM의 EDTA), 열 간단히 75 ° C, 그리고 얼음에 냉각 스냅. 겔의 우물에 전체 샘플을 분배. 또한 비 adenylated 제어 동량 및 저 분자량 크기 표준을 포함​​한다. </li><li> 브롬 페놀 블루는 젤의 바닥에 방법의 ¾ 때까지 24 V / cm에서 젤을 실행합니다. 1X TBE의 1X SYBR 골드 솔루션 장치, 후 얼룩 젤을 분해하여 UV-transilluminator가 상자 (그림 2)에 시각화. NOTE : 입력 RNA와 샘플에 비해 젤 &quot;은 입력 된 RNA는&quot;컨트롤 PCR 산물의 높은 양으로 생성되지 않은 경우는 상술 한 것과 유사한 방식으로 adenylated 올리고 정제. </li></ol></li></ol> 2. 링커 결찰의 miRNA의 3 &#39;끝으로 <ol><li> 3 &#39;결찰 <ol><li> (W / V) PEG 8000, 0.5 및 1.0 μL 50 % : 나열된 순서대로 얼음에 다음과 같은 구성 요소를 조립# 956; 리터 배 RNA 리가 제 완충액 (500 mM 트리스 - 염산 pH를 7.5, 100 mM의의 MgCl 2, 10 mM의 DTT), 1.0 μl의 3 &#39;링커 (100 μM), 0.5 ㎕의 RNA 분해 효소 억제제, 0.5-8.0 μg의 총 RNA를 adenylated 0.5 ㎕의 T4 RNA 리가 제의 2, 5 μL의 최종 부피로 클레아없는 H 2 O. </li><li> 용액 볼텍스에 너무 점성으로 인해 한번 충분히 혼합 PEG 8000의 고농도 피펫로 반응 믹스 가열 뚜껑 열 사이 클러에 반응 장소. 16 ° C에 블록을 설정하고 4 시간 동안 배양한다. 주 : 반응은 결찰 효율 손실없이 20 μL의 최종 볼륨으로 확장 할 수 있습니다. 주위 온도에서 4 시간 동안 반응 또는 밤새 39 ° C에서 본질적으로 동일 결찰 효율을 얻었다. </li></ol></li><li> 젤 정화 <ol><li> 3 &#39;결찰 후 2 배 RNA 로딩 완충액 동량 반응물을 혼합한다. 5 분 동안 70 ° C로 가열하고, 얼음에 멋진 스냅. </li&gt; <li> PAGE 정제를위한 15 %의 폴리 아크릴 아마이드 겔에 샘플을 실행합니다. <ol><li> 단계 150에서 전술 한 것과 유사한 방식으로, 7 M 우레아를 함유하는 15 % 폴리 아크릴 아미드 겔을 준비한다. </li><li> 적어도 30 분 동안 젤 폭의 24 V / cm에서 젤을 사전 실행합니다. 사전 실행과 동일 전압에서 전원 공급 장치, 세척 우물, 부하 샘플 및 실행 젤을 끕니다. </li><li> 샘플을로드하고 브로 모 페놀 블루가 젤을 종료 단지 때까지 페이지를 실행합니다. 목적 생성물이 크실렌 cyanol 가까이 이동한다. </li></ol></li></ol></li><li> 염색, 시각화 및 절제 <ol><li> 15 분 1X 실행 버퍼 (TBE), 1X SYBR 금, 바위와 깨끗한 트레이에 젤 장치 및 장소 젤을 분해. </li><li> 염색 접시에서 젤을 제거하고 깨끗한 플라스틱 랩으로 덮여 자외선 transilluminator가 박스에 시각화. 결찰 제품의 길이는 약 45 뉴클레오티드해야합니다. </li><li> 깨끗한 razo로 결찰 생성물을 함유하는 영역을 절제0.5 ml의 마이크로 원심 분리 관에 R 블레이드 및 장소. </li></ol></li><li> 제품의 분리 및 침전 <ol><li> 피어스 30 G 바늘 0.5 ml의 microcentrifuge 관의 바닥 및 제 깨끗하고 낮은 유지에 피어싱 튜브를 배치, 1.5 ml의 microcentrifuge 관. ~ 15,000 X g에서 5 분 동안 원심 분리기. </li><li> 시각적으로 전체 젤 조각이 내부 튜브의 구멍을 통해 이동 한 것을 확인합니다. 필요한 경우, 가까운 구멍에 약간의 젤 조각을 밀어 깨끗한 피펫 팁을 사용합니다. </li><li> 빈 0.5 ML 튜브를 버리고 아크릴 아미드 슬러리에 높은 소금 열 버퍼 400 μL (25 mM 트리스 - 염산의 pH 7.5, 0.1 % SDS HSCB 400 mM의 염화나트륨을)를 추가합니다. 4 ° C에서 하룻밤 락 튜​​브 (들). </li><li> 넓은 구멍 피펫 팁을 사용하여 0.22 미크론 셀룰로오스 아세테이트 스핀 컬럼에 슬러리를 전송합니다. ~ 15,000 XG에 실온에서 3 분 동안 원심 분리기 멀리 아크릴 아마이드 겔 매트릭스에서 모든 액체를 수집합니다. </li><li> 깨끗하고 낮은 유지에 액체를 이동, 1.5 ml의 튜브 포함20 ㎎ / ㎖ 글리코겐 1 μl를 보내고. 이소프로판올 같은 부피의 1/10 부피의 아세트산 나트륨을 추가한다. 튜브를 수회 반전시켜 용액을 혼합하고 20 분 동안 냉동실 ° C에서 -80 침전. </li><li> 20 분 동안 4 ° C에서 최고 속도로 원심 분리기는 핵산을 펠렛. 상층 액을 제거하고 70 % 에탄올로 씻는다. 공기 건조 실온에서 10 분 동안 펠릿 5 &#39;결찰 단계로 바로 진행. </li></ol></li></ol> 링커 하이브리드 3. 링커 내고 5 &#39;의 miRNA-3의 끝&#39; <ol><li> 펠렛 핵산, 다음과 같은 구성 요소 추가 : 2 μL PEG 8000 (50 % / W V), 0.5 μL 10 배 RNA 리가 버퍼, 0.5 μL의 ATP (10 mM)을 0.5 μL NN-RNA 올리고 (100 μM) 및 H 4 ㎕의 최종 부피로 2 O. NOTE : 5&#39;-반전 디데 옥시 TCUACAGUCCGACGAUCNN3 다음 &#39;와 제조사에서 HPLC를 통해 정제시켜 같이&#39;NN-RNA 올리고 &#39;의 조성물이다. </li><li> 혼합아래로 반복적으로 피펫 팅에 의해이 샘플. 펠렛 완전히 다시 용해되어 있는지 확인하는 데 시간 (최대 5 분까지)에 대해이 작업을 수행합니다. 다시 용해가 문제가있는 경우 37 ° C에 샘플 잠시 (3-5 분) 가열 혼합 피펫을 다시 시작합니다. </li><li> RNA 분해 효소 억제제 및 T4 RNA 리가 제 1 (5 대)의 1 : 1 혼합물 펠릿을 용액에 다시되면, (1)의 1 μl를 함유하는 깨끗한 PCR 튜브에 콘텐츠를 전송. 다시 피펫 아래로 섞는다. </li><li> 4 시간 동안 37 ° C에서 열 사이 클러에 반응을 부화. cDNA 합성에 바로 진행합니다. </li></ol> 4. 역전사 / cDNA를 합성 <ol><li> 5 다음과 같은 구성 요소를 추가 &#39;결찰 반응 : 2 μL 5 배 RT 버퍼, 0.75 μL 100 mM의 DTT, 1 μL 일루미나 RT 프라이머 5&#39;-GCCTTGGCACCCGAGAATTCCA-3&#39;(1 μM) 0.5 μL의 dNTPs를 (10 mm)이다. </li><li> 5 분 동안 65 ° C로 피펫과 열에 의해 섞는다. 그런 다음 천천히 벤치 t에 냉각영업 이익. </li><li> 반응물을 얼음에 실온, 도시 튜브로 냉각시키고, 역전사 효소 및 RNA 분해 효소 억제제의 각 0.5 μl를 추가하면. 제조업체의 지침에 표시된 확장 단계에 대한 열 자전거 타는 사람에 피펫 팅 장소로 섞는다. </li><li> 완료되면 추가는 핵산 분해 효소가없는 H 2 O 5 μL 15 μL까지의 cDNA 라이브러리의 최종 볼륨을 가지고 있습니다. </li></ol> 5. 도서관 준비 <ol><li> 인덱싱 PCR를 들어, 얼음에 다음과 같은 반응 조립 : 6 μL 5 배 Phusion 버퍼, 1 μL의 dNTPs를 (10 mM)을 0.9 μL DMSO, 0.75 μL 5 &#39;프라이머 (25 μM), 0.75 μL 3&#39;프라이머 (25 μM) 3 μL의 cDNA 라이브러리 (입력의 양에 따라 가변 농도), 0.5 ㎕의 폴리머 라제 Phusion (1 부), 30 ㎕의 최종 부피로 클레아없는 H 2 O. 템플릿과의 시퀀싱 플랫폼과의 호환성을 보장하기 위해 프라이머 시퀀스를 선택선택. <ol><li> 15 초 동안 72 ° C에서 연장, 제 4 사이클, 3 분 동안 98 ° C에서 초기 변성을 15 초 동안 50 ° C에서 15 초, 어닐링 98 ° C 변성 : PCR을 다음과 같이 설정하여 반응을 실행 . 주 : 입력 재료의 양에 따라, 전체 PCR 사이클 수를 변화. 통상적으로, 총 RNA의 1-2 μg의 입력, 총 13 PCR 사이클은 miR-SEQ 라이브러리를 생성하기에 충분하다. </li><li> 어닐링 및 연장 사이클 합하고 30 초 및 변성을 위해 70 ° C에서 수행되는 두 단계의 방법으로 예를 들어 후속 사이클, 5-12 사이클을 수행해 다시 15 초 동안 98 ° C이다. NOTE은 : 반응 부피는 PCR 프로그램을 일시 정지 허가 및 목적 생성물의 출현을 모니터링하기 위해 미리 결정된 사이클 번호에 10 μl의 분취 량을 제거하기 위해 30 μL이다. 일반적으로, 사이클 10, 13, 및 16은 총 RNA 샘플로부터의 시험을 위해 선택된다. </li></ol></li><li> 준비40 % 아크릴 아미드 2 ㎖, 10 배 TBE 1 ㎖, 및 H 2 O 10 ㎖에 혼합하여 8 % 비 - 변성 아크릴 아마이드 겔. 그런 다음 10 % APS 100 ㎕ 및 TEMED의 10 μl를 추가합니다. 젤을 붓고 설정할 수 있습니다. <ol><li> 30 분 동안 젤 폭의 12 V / cm에서 젤을 사전 실행합니다. 세척 우물. 10 배 로딩 버퍼 1 μL 추가 (15 % 피콜-400, 66 mM의 EDTA, 20 mM 트리스 - 염산의 pH 8.0, 0.1 % (w / 블루 / V) 브로 모 페놀 승 V) SDS, 0.01 % (), 10 μl를 추가 치수의 각 웰 5mm X 1mm X 15mm에 샘플. 일반적으로, 각 웰 샘플 30 μL까지 넣을 수 있습니다. 브롬 페놀 블루 염료가 바로 젤의 바닥에서 실행될 때까지 1X TBE 버퍼에 젤 폭의 10 V / cm에서 샘플 젤을 실행합니다. </li><li> 얼룩 단계 2.3에서 기술 된 바와 같이 겔을 시각화. </li><li> 146 염기쌍 밴드를 절제하고 2.4 절에 설명되어있는 것과 유사한 방식으로 HSCB 0.25 ml의 하룻밤 용출. 이소프로판올 같은 부피의 1/10 부피 아세트산 나트륨에서 DNA를 침전. 70 % 전자에 세척 펠렛TE 버퍼에 thanol 및 재현 탁. </li><li> 제조업체의 권장 사항에 따라 picogreen 사용하여 라이브러리를 정량화. </li></ol></li></ol>

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V., 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=Limitations+and+possibilities+of+small+RNA+digital+gene+expression+profiling.">Limitations and possibilities of small RNA digital gene expression profiling.</a> Nature Methods. 6 (7), 474-476 (2009).</li><li>Jayaprakash, A. D., Jabado, O., Brown, B. D., Sachidanandam, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+remediation+of+biases+in+the+activity+of+RNA+ligases+in+small-RNA+deep+sequencing.">Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing.</a> Nucleic Acids Research. 39 (21), 141 (2011).</li><li>McLaughlin, L. W., Romaniuk, E., Romaniuk, P. J., Neilson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Acceptor+Oligoribonucleotide+Sequence+on+the+T4+RNA+Ligase+Reaction.">The Effect of Acceptor Oligoribonucleotide Sequence on the T4 RNA Ligase Reaction.</a> European Journal of Biochemistry. 125 (3), 639-643 (1982).</li><li>Zhuang, F., Fuchs, R. T., Sun, Z., Zheng, Y., Robb, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bias+in+T4+RNA+ligase-mediated+3&#8242;-adapter+ligation.">Structural bias in T4 RNA ligase-mediated 3&#8242;-adapter ligation.</a> Nucleic Acids Research. 40 (7), 54 (2012).</li><li>Harrison, B., Zimmerman, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer-stimulated+ligation:+enhanced+ligation+of+oligo-+and+polynucleotides+by+T4+RNA+ligase+in+polymer+solutions.">Polymer-stimulated ligation: enhanced ligation of oligo- and polynucleotides by T4 RNA ligase in polymer solutions.</a> Nucleic Acids Research. 12 (21), 8235-8251 (1984).</li><li>Torchia, C., Takagi, Y., Ho, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archaeal+RNA+ligase+is+a+homodimeric+protein+that+catalyzes+intramolecular+ligation+of+single-stranded+RNA+and+DNA.">Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.</a> Nucleic Acids Research. 36 (19), 6218-6227 (2008).</li><li>Lau, N. C., Lim, L. P., Weinstein, E. G., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Abundant+Class+of+Tiny+RNAs+with+Probable+Regulatory+Roles+in+Caenorhabditis+elegans.">An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans.</a> Science. 294 (5543), 858-862 (2001).</li><li>Yi, R., 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=DGCR8-dependent+microRNA+biogenesis+is+essential+for+skin+development.">DGCR8-dependent microRNA biogenesis is essential for skin development.</a> Proceedings of the National Academy of Sciences. 106 (2), 498-502 (2009).</li><li>Leung, A. K. L., 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=Genome-wide+identification+of+Ago2+binding+sites+from+mouse+embryonic+stem+cells+with+and+without+mature+microRNAs.">Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs.</a> Nature Structural &amp; Molecular Biology. 18 (2), 237-244 (2011).</li></ol>

The authors declare no competing financial interests.

본원에 기재된 방법은 결찰 효율, 즉 PEG, 무작위 링커의 사용의 높은 농도, 및 링커 2,6,9 고농도을 최대화하기 위해 여러 주요 변수를 사용한다. 이 방식은 총 RNA 시료 (2)로부터 안정적으로 정량적 시퀀싱 라이브러리를 허용한다. 우리는 입력 RNA의 다수의 적정을 수행하고 선행하는 방법론 (데이터 미기재) 1-8 μg의 범위에서 총 RNA 량에 가장 적합한 것으로 결론 지었다. 10-500 ng의 범...

높은 처리량 시퀀싱 기반의 방법론은 널리 크게 생물학적 시스템 3,4의 분자 복잡성에 대한 우리의 이해를 확장 최근 몇 년 동안 많은 생물학적 시료에 적용되고있다. 그러나, 높은 처리량 시퀀싱 RNA 샘플의 제조는 종종 이러한 강력한 기술의 잠재 성을 제한하는 고용 방법론에 내재 특정 바이어스를 부여한다. 이 방법은 특정 편견 잘 결찰 기반, 작은 RNA 라이브러리 준비 1,2,5,6- 문서화되어있다. 이러한 편견은 1,000 배의 변화에​​에서 격렬 변수 및 오류가 발생하기 쉬운 시퀀싱 데이터에서 miRNA의 풍요의 추론을, 몰 합성의 miRNA에 대한 숫자를 읽 결과. 파아지 - 유래의 T4 RNA 리가 제의 특성에 초점을 연구 효소는 높은 처리량 시퀀싱 실험에서와 같이 편향된 라이브러리를 나타내 뉴클레오타이드 기반 선호도 7 나타내는 것을 문서화 <s1,2,8&gt;까지. RNA 리가 제에 의해 부여 된 편향을 최소화하기 위하여, 여러 전략이 사용되었다; 거대 분자, 9 군집 결찰 사이트 6 근접한 어댑터 뉴클레오타이드 서열을 랜덤 화하고, 결찰 어댑터 (2)의 높은 농도를 채용. 이 세 가지 방법의 조합을 통해 우리는 높은 처리량 시퀀싱 (그림 1) 호환 작은 RNA 라이브러리의 편견 준비를위한 작업 흐름을 개발했다. 현재의 프로토콜과 우리의 최적화 된 방법 사이의 직접 비교를 위해, 우리의 최근 보고서 2를 참조하시기 바랍니다. 이 최적화 된 방법은 모두 3 &#39;및 5&#39;단계에서 95 % 초과의 결찰 효율을 산출하고, 합성과 생물학적 시료 (2)로부터 소형 RNA 분자의 공평 결찰을 허용한다.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Reagent/ Equipment</td>
			<td style="width:175px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:280px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">3&#39; Linker (5&#39; phosphorylated, 3&#39; blocked)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">5&#39; Linker</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>5&#39; blocked, HPLC Purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL2 (1-249 K227Q)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0351S</td>
			<td>Specialized for ligation of pre-adenylated DNA adapters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (without ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">10X Ligation Buffer (with ATP)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">RNaseOUT&#160;</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Polyethylene Glycol (mol. Wt. 8000)</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Nuclease-free water</td>
			<td style="width:175px;">Ambion</td>
			<td style="width:157px;">AM9937</td>
			<td>We have found water collected from a distillation apparatus to be of equvalent quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">T4RNL1</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0204L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Superscript III RT kit</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">18080-051&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phusion PCR kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">M0530S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RP1 Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina RT Primer</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:261px;">Illumina Index Primer(s)</td>
			<td style="width:175px;">Integrated DNA Technologies</td>
			<td style="width:157px;">custom</td>
			<td>Sequence information available from Illumina</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">40% Acrylamide</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">BP14081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Urea</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">U6504</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ammonium persulfate</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tetramethyethylenediamine (TEMED)</td>
			<td style="width:175px;">Sigma Aldrich</td>
			<td style="width:157px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">2X Denaturing RNA loading buffer</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">Included with M0351S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Razor blades</td>
			<td style="width:175px;">VWR</td>
			<td style="width:157px;">55411-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SpinX Centricon Tubes</td>
			<td style="width:175px;">Costar</td>
			<td style="width:157px;">CLS8161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Low Retention Microfuge tubes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:157px;">02-681-320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sybr Gold</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:157px;">S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Adenylation Kit</td>
			<td style="width:175px;">New England Biolabs</td>
			<td style="width:157px;">E2610L</td>
			<td></td>
		</tr>
	</tbody>
</table>

highly efficient ligation of small rna molecules for microrna quantitation by high-throughput sequencing

미르 복제 및 높은 처리량 시퀀싱 미르-SEQ는, 단일 염기 해상도의 miRNAs를 정량화하기 위해 사체 차원의 접근 방식으로 독립적이라고. 이 기술은 miRNA의 분자 3 &#39;, 5&#39;올리고 뉴클레오티드 어댑터를 연결하여 miRNA가를 캡처하고 새로이 miRNA의 검색을 할 수 있습니다. 강력한 차세대 시퀀싱 플랫폼과 커플 링,은 miR-SEQ는 miRNA의 생물학의 연구 수단이되어왔다. 그러나, 올리고 뉴클레오티드 결찰 단계에 의해 도입 상당한 편향 정확한 정량 도구로서 사용되는은 miR-SEQ을 방지했다. 이전의 연구는 현재의 miR-SEQ 방법의 편견은 종종 일부 miRNA가 1,2 1,000 배까지 오류가 부정확 한 miRNA의 정량으로 이어질 것을 보여줍니다. RNA 결찰에 의해 부여 이러한 편견을 해결하기 위해, 우리는 모두 3 &#39;, 5&#39;결찰 단계에 대한 95 %의 결찰 효율성을 초래 작은 RNA 결찰 방법을 개발했다. 이 메신저를 벤치마킹지속적으로 기대 값에서보다 두 배 편차 번호를 읽어 산출, 몰 또는 차등 혼합 된 합성의 miRNA를 사용하여 라이브러리 생성 방법을 입증했다. 또한,이 고효율의 miR-SEQ 방법은 생체 내 총 RNA 샘플 2에서 정확한 게놈 전체의 miRNA의 프로파일 링을 허용한다.

위의 방법에 대한 예상 결과는 처음에 (그림 2) M의 RNA 리가 아제에 의한 adenylation의 대상이었다 DNA 올리고 뉴클레오타이드의 크기의 단일 염기 변화 (증가)의 관찰해야한다. 3 &#39;결찰 후, 아크릴 아미드 겔의 시각화 (도 3 참조) 겔 100-300 뉴클레오티드 영역에서 분명 날카로운 고 분자량 밴드를 나타낸다. 이것은 사용되는 총 RNA 샘플 고품질 인 것을 나타낸다 (악화?...

Watch this Scientific Journal Video about Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing at JoVE.com

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

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 ...

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Biology

11.8K 조회수.  University of Colorado, Boulder. 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. 

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

Gramicidin-based Fluorescence Assay; for Determining Small Molecules Potential for Modifying Lipid Bilayer Properties

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions. Changes in bilayer properties thus alter membrane protein function, which provides an indirect way for amphiphiles to modulate protein function and a possible mechanism for "off-target" drug effects. We have previously developed an electrophysiological assay for detecting changes in lipid bilayer properties using linear gramicidin channels as probes 3,12. Gramicidin channels are mini-proteins formed by the transbilayer dimerization of two non-conducting subunits. They are sensitive to changes in their membrane environment, which makes them powerful probes for monitoring changes in lipid bilayer properties as sensed by bilayer spanning proteins. We now demonstrate a fluorescence assay for detecting changes in bilayer properties using the same channels as probes. The assay is based on measuring the time-course of fluorescence quenching from fluorophore-loaded large unilamellar vesicles due to the entry of a quencher through the gramicidin channels. We use the fluorescence indicator/quencher pair 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)/Tl+ that has been successfully used in other fluorescence quenching assays 5,13. Tl+ permeates the lipid bilayer slowly 8 but passes readily through conducting gramicidin channels 1,14. The method is scalable and suitable for both mechanistic studies and high-throughput screening of small molecules for bilayer-perturbing, and potential "off-target", effects. We find that results using this method are in good agreement with previous electrophysiological results 12.

We introduce a fast fluorescence-based assay that monitors the rate of fluorescence quenching as a measure of gramicidin channel activity. The gramicidin channels are used as molecular force transducers to monitor changes in lipid bilayer properties as sensed by bilayer spanning proteins.

그램이시딘 기반의 형광 분석, 지질 이중층의 속성을 수정하는 작은 분자 잠재 결정

Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter ...

연구

생물학

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

안쪽 정류기의 칼륨 채널의 소형 분자 변조기에 대한 높은 처리량 검사

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / 카스 시스템의 Endonucleases을위한 기판 생성

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

높은 처리량 SHAPE를 사용하여 RNA 이차 구조 예측

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

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 ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

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 ...

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

자동화 모​​듈 높은 처리량 엑소 폴리 사카 라이드 심사 플랫폼은 매우 민감한 탄수화물 지문 분석과 결합

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food ...

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 ...

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 ...