This protocol was developed in support of a Joint Genome Institute-Community Sequencing Project (#1657). The work was supported in part by the Philomathia Foundation, the National Science Foundation (#0929226) and the Energy Biosciences Institute to S. Somerville; and by Swiss National Science Foundation (#310030_163260) to B. Keller. We would like to thank our colleagues, M. Figureroa and M. Miller (University of Minnesota), R. Panstruga (RWTH Aachen University), C. Pedersen (University of Copenhagen), P. Spanu (Imperial College), J. Taneja (University of California Berkeley), M. Wildermuth (University of California Berkeley), R. Wise (Iowa State University) and S. Xiao (University of Maryland) for their generous advice and for volunteering their protocols as we developed the protocol described here.

곰팡이 재료 1. 준비 <ol><li> 흰가루병 성장 <ol><li> 다음의 조건으로 성장 챔버에 식물을 성장 : 22 ° C 일 온도 20 ℃ 밤 온도, 80 % 상대 습도, 14 시간 낮 길이 125 μE / m 2 / s의 광 강도 (형광등에 의해 제공된 ). </li><li> 18, 29 설명 Golovinomyces cichoracearum 변형 UCSC1 오이 식물 (쿠쿠 미스 사티의 다양한 부시 챔피언) 감염. </li></ol></li><li> 수확 흰가루병 분생 포자 <ol><li> 생포 축적 된 인 - 라인 필터 (11 μm의)와 작은 진공을 이용하여 접종 10 일 - 7에 감염된 식물을 수확 분생. 부드러운 압력을 적용, 분생 포자를 수집하는 잎의 표면을 따라 진공 노즐을 실행합니다. 참고 : G.의 cichoracearum의 평균 수익률은 크게 infe에서 분생 포자의 20 밀리그램이다면적이 약 130cm 2 cted 오이 잎. </li><li> 깨끗한 종이 상에 필터로부터 (7-8 감염된 오이 잎으로부터 약 375 μL 부피 또는 분생) 분생 포자 약 150 mg의 브러시. 여기에서, (액체 질소)에서 미리 냉각 두 32분의 5 인치 직경의 스틸 밀링 볼 2 mL의 크리오 바이알 (cryovial)의 분생 포자로 옮긴다. 즉시 액체 질소로 튜브를 반환한다. -80 ° C에서 보관 튜브. </li></ol></li><li> 볼 밀링에 의해 속보 열기 분생 포자 <ol><li> 플래시를 액체 질소 중에서 볼밀 알루미늄 판을 고정. 로드는 볼 밀 랙에 크리오 바이알 (cryovial). 격렬 (실온) 30 회 / 초에서 30 초 동안 볼 밀에서 분생 스틸 볼과 크리오 바이알 (cryovial)을 흔들어. 즉시 액체 질소에서 재 냉동을 크리오 바이알 (cryovial). 참고 : 처음에, 연마하는 동안 세포 벽 파열의 효율을 100 배 배율에서 현미경으로 분생 포자의 샘플을 액세스 할 수 있습니다. 30 회 / 초, B 30의 2 라운드 - 필요한 경우, 추가로 1 갈기유타는 최소로 라운드의 수를 유지한다. 최적의 조건이 성립 된 후,이 단계에서 현미경으로 포자 파손 평가 루틴은 불필요하다. </li></ol></li></ol> 2. 게놈 DNA 정제 <ol><li> 분생 포자 용해 <ol><li> 빨리 작업, 해동 분생 포자를 방지 자석 크리오 바이알 (cryovial)에서 강구를 제거합니다. 슬러리가 형성 될 때까지 10 초 - 65 μL 700 ° C의 용해 완충액 (표 1) 및 선회 (5)를 추가한다. </li><li> 300 μL (65 °의 C) 예열 된 5 % (v / v)의 sarcosyl 추가 부드럽게 섞어 다섯 번 (3 초 당 1 개 반전) 반전. 65 ° C에서 30 분 동안 인큐베이션 튜브; 10, 20, 30 분에서 3 시간 온화하게 반전. 넓은 구멍 피펫 팁을 사용하여, 새로운 2 ML의 마이크로 원심 튜브에 전 용액을 옮긴다. 이후의 모든 단계에서, 부드럽게, 천천히 반전에 의해 혼합 넓은 구멍 피펫 팁으로 DNA 함유 솔루션을 전송합니다. </li></ol></li><li> DNA 추출 I <ol><li> 클로로의 1 볼륨을 추가성상 : 이소 아밀 알코올 : 부드럽게 용해 용액 (24 1 V / V)과 5 회 혼합 바꾸. 부드럽게 배양의 끝에서 다시 중간 지점에서 다섯 번을 혼합하는 반전, 실온에서 10 분 동안 품어. 14,000 × g에서 15 분 동안 실온에서 원심 분리기. 넓은 구멍 피펫 팁을 사용하여 조심스럽게 새로운 2 ML의 마이크로 원심 튜브에 수성층을 전송; 계면으로부터 재료를 포함하는 피. </li></ol></li><li> DNA 강수량 I <ol><li> 1 볼륨 실온 100 % 이소프로판올 (약 750 μL)을 추가하고 부드럽게 여섯 번 섞어 반전. 14,000 × g에서 15 분 동안 실온에서 원심 분리기. 조심스럽게 상층 액을 제거하고 폐기합니다. </li><li> 450 μL 사전 냉장 (-20 ° C) 70 % 에탄올을 추가합니다. 14,000 × g으로 실온에서 5 분 동안 원심 분리기. 조심스럽게 상층 액을 제거하고 폐기합니다. 조심스럽게 미세 피펫 팁으로 뜨는을 제거하고 폐기, 3 초 동안 1,000 XG에서 원심 분리기. 15 분 A의 펠릿을 공기 - 건조t 실온. 15 분 이상 건조하지 마십시오. </li><li> 300 μL TE의 펠릿 (10 mM 트리스 - (CL), 1mM의 에틸렌 디아민 테트라 아세트산의 pH 8.0)에 재현 탁. 4 ℃에서 밤새 인큐베이션. 부드럽게 솔루션에 가지 않았다 잔여 물질을 재 부유 버려. </li></ol></li><li> , RNA 오염 제거 10 μL의 RNase A (10 ㎎ / ㎖)을 첨가한다. 부드럽게 세 번을 섞어 반전. 3 초 동안 1,000 XG에서 원심 분리기. 2 시간 동안 37 ℃에서 인큐베이션. </li><li> DNA 추출 II <ol><li> 300 μL를 페놀 추가 : 클로로포름 : 이소 아밀 알코올 (25 : 24 : 1 v / v)로 부드럽게 다섯 번을 섞어 반전. 부드럽게 배양의 끝에서 다시 중간 마크에서 다섯 번을 혼합하는 반전, 실온에서 10 분을 품어. 14,000 × g으로 실온에서 15 분간 원심 분리기. 넓은 구멍 피펫 팁을 사용하여, 새로운 1.5 ㎖의 마이크로 원심 튜브에 상기 상층 액을 전송. </li></ol></li><li> DNA 강수량 II <ol><li> 0.01 부피 3 M 아세트산 나트륨 pH를 5.2 (approximatel 추가Y 3 μL), 부드럽게 다섯 번을 섞어 반전. 2.5 볼륨 사전 냉장 (-20 ° C) 100 % 에탄올 (약 750 μL)를 추가합니다. 부드럽게 다섯 번을 섞어 반전. -20 ℃에서 하룻밤 품어 </li><li> 원심 분리기에서 14,000 × g에서 4 ℃에서 30 분. 조심스럽게 상층 액을 제거하고 폐기합니다. </li><li> 450 μL 사전 냉장 (-20 ° C) 70 % 에탄올을 추가합니다. 14,000 × g에서 4 ℃에서 5 분 동안 원심 분리기. 조심스럽게 상층 액을 제거하고 폐기합니다. 3 초 동안 1,000 XG에서 원심 분리기. 조심스럽게 미세 피펫 팁 및 폐기에 뜨는을 제거합니다. </li><li> 실온에서 30-60 분 동안 펠렛을 공기 - 건조. 27.5 μL TE의 펠렛을 재현 탁. 4 ℃에서 밤새 인큐베이션. 부드럽게 솔루션에 가지 않았다 잔여 물질을 재 부유 버려. </li><li> 분취 품질 관리 시험 22.5 μL TE (최종 부피 25 μL)에 2.5 μL (아래 참조). 시퀀싱을 위해 제출 될 때까지 4 ℃에서 저장 DNA 샘플. 장기 보관, 저장 샘플의 경우및 -80 ° C에서 전단을 방지하기 위해 동결 - 해동 사이클을 최소화 할 수 있습니다. 참고 : 제대로 피펫 어려울 수있다 고 분자량 게놈 DNA로이 단계에서 피펫 팅 할 때주의하십시오, &quot;QC 나누어은&quot;게놈 DNA 샘플의 정확한 표현인지 확인하는 것이 중요하다. </li></ol></li></ol> 3. 품질 관리 <ol><li> 샘플 순도 <ol><li> TE에 빈을 사용하여, 작은 볼륨 분광 광도계에 QC 나누어지는 1 μL를로드하여 DNA의 샘플 순도를 평가합니다. 기록 A260 / A280 및 A260 / A230 수치. 순수한 DNA는 1.8과 2.2 사이에 약 1.8 A260 / A230의 비율 A260 / A280 비율을 가질 것이다. </li></ol></li><li> 게놈 DNA의 정량화 <ol><li> 제조업체의 지침에 따라 상업 이중 가닥 DNA 정량 분석 ​​키트를 사용하여 품질 관리 나누어의 정량화를 수행합니다. </li></ol></li><li> DNA 품질 평가 I <ol><li> 부하 약 60 ng의 게놈 DNA 및1X TAE (40 개 mM 트리스, 20 mM의 아세트산 및 1 mM의 EDTA, pH를 8.0)에 0.7 % 아가 로즈 겔상에서 DNA 사다리. 70 V 40 겔 실행 장치 - 게놈 DNA 밴드까지 45 분 정도 사다리에서 잘 분리 대역에서 2cm이다 명백하다. 이러한 조건은 가능한 겔 전기 영동 장치에 기초하여 조절 될 필요가있다. </li></ol></li><li> DNA 품질 평가 II <ol><li> 부하는 약 60 0.5X의 TBE (44.5 mM 트리스, 445 mM의 붕산, 1 mM의 EDTA, pH를 8.3) 및 1 % 아가 로스 겔상에서 게놈 DNA 및 (펄스 필드 겔 전기 이동에 적합) 고 분자량 DNA 사다리 겨 16 시간 동안 80 KBP 파형 펄스 필드 겔 전기 프로토콜 - 5을 사용하여 0.5X TBE를 실행하는 버퍼에서 실행. </li></ol></li><li> 세균, 곰팡이 및 공장 오염 물질의 평가 <ol><li> 프라이머 및 증폭 조건을 이용리스트 리보솜 유전자의 적절한 전사 내부 스페이서 (ITS) 영역을 증폭하는 중합 효소 연쇄 반응 (PCR)을 수행표 2에 에드. 겔에서 얻어진 반응 하중 10 μL. 참고 : ITS 지역 ITS 프라이머 곰팡이로 증폭해야 흰가루병을 나타내는 밴드. 이 앰플 리콘 및 세균 또는 식물 프라이머로부터 생성 된 앰플 리콘 DNA는 오염의 근원을 결정하기 위해 서열화한다. 만 흰가루병은 ITS 시퀀스는 곰팡이의 증폭에서 찾을 수 있습니다. </li></ol></li></ol>

<ol>
	<li>Agrios, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Pathology. , (1969).</li><li>Braun, U., Cook, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Taxonomic manual of the Erysiphales (Powdery Mildews). , (2012).</li><li>Both, M., Csukai, M., Stumpf, M. P. H., Spanu, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+Blumeria+graminis+indicate+dynamic+changes+to+primary+metabolism+during+development+of+an+obligate+biotrophic+pathogen.">Gene expression profiles of Blumeria graminis indicate dynamic changes to primary metabolism during development of an obligate biotrophic pathogen.</a> Plant Cell. 17 (7), 2107-2122 (2005).</li><li>Glawe, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+powdery+mildews:+A+review+of+the+world's+most+familiar+(yet+poorly+known)+plant+pathogens.">The powdery mildews: A review of the world's most familiar (yet poorly known) plant pathogens.</a> Annu Rev Phytopathol. 46, 27-51 (2008).</li><li>Fabro, G., 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+expression+profiling+Arabidopsis+at+the+stage+of+Golovinomyces+cichoracearum+haustorium+formation.">Genome-wide expression profiling Arabidopsis at the stage of Golovinomyces cichoracearum haustorium formation.</a> Plant Physiol. 146 (3), 1421-1439 (2008).</li><li>H&#252;ckelhoven, R., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+the+plant-powdery+mildew+interaction.">Cell biology of the plant-powdery mildew interaction.</a> Curr Opin Plant Biol. 14 (6), 738-746 (2011).</li><li>Micali, C. O., Neumann, U., Grunewald, D., Panstruga, R., O'Connell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+a+specialized+plant-fungal+interface+during+host+cell+internalization+of+Golovinomyces+orontii+haustoria.">Biogenesis of a specialized plant-fungal interface during host cell internalization of Golovinomyces orontii haustoria.</a> Cell Microbiol. 13 (2), 210-226 (2011).</li><li>Chandran, D., Inada, N., Hather, G., Kleindt, C. K., Wildermuth, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+Arabidopsis+cells+at+the+powdery+mildew+infection+site+reveals+site-specific+processes+and+regulators.">Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators.</a> Proc Natl Acad Sci. 107 (1), 460-465 (2010).</li><li>Spanu, P. D., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Powdery+mildew+genomes+in+the+crosshairs.">Powdery mildew genomes in the crosshairs.</a> New Phytol. 195 (1), 20-22 (2012).</li><li>Vela-Corc&#237;a, D., Romero, D., Tor&#233;s, J. A., De Vicente, A., P&#233;rez-Garc&#237;a, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+Podosphaera+xanthii+by+electroporation+of+conidia.">Transient transformation of Podosphaera xanthii by electroporation of conidia.</a> BMC Microbiol. 15 (20), (2015).</li><li>Hacquard, S., Francis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Botanical Research. 70, 109-142 (2014).</li><li>Spanu, P. D., 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+expansion+and+gene+loss+in+powdery+mildew+fungi+reveal+tradeoffs+in+extreme+parasitism.">Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.</a> Science. 330 (6010), 1543-1546 (2010).</li><li>Wicker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wheat+powdery+mildew+genome+shows+the+unique+evolution+of+an+obligate+biotroph.">The wheat powdery mildew genome shows the unique evolution of an obligate biotroph.</a> Nat Genet. 45 (9), 1092 (2013).</li><li>Jones, 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=Adaptive+genomic+structural+variation+in+the+grape+powdery+mildew+pathogen,+Erysiphe+necator.">Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator.</a> BMC Genomics. 15, 1081 (2014).</li><li>Thomma, B. P. H. J., 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=Mind+the+gap;+seven+reasons+to+close+fragmented+genome+assemblies.">Mind the gap; seven reasons to close fragmented genome assemblies.</a> Fungal Genet Biol. 90, 24-30 (2016).</li><li>Parlange, F., 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=A+major+invasion+of+transposable+elements+accounts+for+the+large+size+of+the+Blumeria+graminis+f.sp.+tritici+genome.">A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome.</a> Funct Integr Genomics. 11, 671-677 (2011).</li><li>Faino, 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=Single-molecule+real-time+sequencing+combined+with+optical+mapping+yields+completely+finished+fungal+genome.">Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome.</a> mBio. 6, (2015).</li><li>Adam, 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=Comparison+of+Erysiphe+cichoracearum+and+E.+cruciferarum+and+a+survey+of+360+Arabidopsis+thaliana+accessions+for+resistance+to+these+two+powdery+mildew+pathogens.">Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens.</a> Mol Plant Microbe In. 12 (12), 1031-1043 (1999).</li><li>Bourras, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+avirulence+loci+and+allele-specific+effector+recognition+control+the+PM3+race-specific+resistance+of+wheat+to+powdery+mildew.">Multiple avirulence loci and allele-specific effector recognition control the PM3 race-specific resistance of wheat to powdery mildew.</a> Plant Cell. 27 (10), 2991-3012 (2015).</li><li>Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant genotyping II: SNP technology. , (2008).</li><li>Healey, A., Furtado, A., Cooper, T., Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+a+simple+method+for+extracting+next-generation+sequencing+quality+genomic+DNA+from+recalcitrant+plant+species.">Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.</a> Plant Methods. 10 (21), (2014).</li><li>Fang, G., Hammar, S., Grumet, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+inexpensive+method+for+removing+polysaccharides+from+plant+genomic+DNA.">A quick and inexpensive method for removing polysaccharides from plant genomic DNA.</a> Biotechniques. 13 (1), 52-54 (1992).</li><li>Jordon-Thaden, I. E., Chanderbali, A. S., Gitzendanner, M. A., Soltis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+CTAB+and+TRIzol+protocols+improve+RNA+extraction+from+chemically+complex+Embryophyta.">Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.</a> Appl Plant Sci. 3 (5), 1400105 (2015).</li><li>Murray, M. G., Thompson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+high+molecular+weight+plant+DNA.">Rapid isolation of high molecular weight plant DNA.</a> Nucleic Acids Res. 8 (19), 4321-4325 (1980).</li><li>Greco, M., S&#225;ez, C. A., Brown, M. T., Bitonti, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+effective+method+for+high+quality+co-extraction+of+genomic+DNA+and+total+RNA+from+low+biomass+Ectocarpus+siliculosus,+the+model+brown+alga.">A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.</a> PLoS One. 9 (5), e96470 (2014).</li><li>Wilson, I. W., Schiff, C. L., Hughes, D. E., Somerville, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+analysis+of+powdery+mildew+disease+resistance+in+the+Arabidopsis+thaliana+accession+Kashmir-1.">Quantitative trait loci analysis of powdery mildew disease resistance in the Arabidopsis thaliana accession Kashmir-1.</a> Genetics. 158 (3), 1301-1309 (2001).</li></ol>

The authors have nothing to disclose.

가지 의무적 biotrophic 흰가루병 균 순수한 고 분자량의 게놈 DNA를 얻기 위해, 전술 한 방법의 변형 된 버전 (30)를 개발 하였다. 이 최적의 프로토콜을 이용하여 평균 수율은 150 mg의 분생 당 7 μg의 DNA를, 종래의 프로토콜로 얻어진 수율의 두배이다. 또한, 평균 크기는 48.5 KBP에 걸쳐 약 20 KBP에서 증가했다. 이 프로토콜은 cucumber- G.의 cichoracearum 시스템에 최적화되었다. biotrophic...

<html> 흰가루병은 가지 의무적 biotrophic 곰팡이 식물의 그룹이 함께 할 때, 식물 병 전 세계적으로 하나의 가장 큰 원인, 즉 병원균이다. 분류 학적으로 가족 Erisyphaceae 2에서 다섯 개 종족으로 분류 된 흰가루병의 900 설명 종있다. 경제적 중요성과 그들의 호스트와 개발 친밀한 관계를 모두 인해, 흰가루병 질환&gt; 100 년 연구의 대상이되어왔다. 감염시, 흰가루병이 병원균 혜택을, 세포의 구조, 신진 대사와 그 호스트의 분자 생물학의 급격한 변화를 유도. 그러나, 흰가루병의 연구는 그들의 가지 의무적 라이프 스타일, 그리고 순수 문화에 곰팡이의 성장은 아직 5, 4, 3에 설명되지 않은에 특히 도전,&quot;외부 참조&quot;&gt; 6, 7, 8. 과도 변환이 어떤 종 9, 10에보고되었지만 흰가루병의 신뢰성, 안정적인 유전자 변형은 아직 완성되지 않았습니다. 흰가루병 게놈의 염기 서열 및 조립으로 인해 게놈 자체의 기능들이 어려운 입증했다. 다른 진균 유전체로 - (180 MBP 120) 기준으로 60 구성 - 흰가루병 게놈이 크고 90 % 고르게 분포 반복 소자 (11). 이러한 요소는 비 긴 말단 반복뿐만 아니라 분류 중복 요소를 포함한다. 하나의 흰가루병 종, Blumeria 그라 미니 f를 두 formae SPECIALES. SP. hordei 및 F. SP. 소맥 (년 Bgh 각각 BGT)뿐만 아니라 포도 흰가루병에 에리시 페 necator, 꿀벌이N 서열, 및 다른 여러위한 초안 게놈은 12, 13, 14을 완성 하였다. 게놈의 반복적 인 특성은 조립 어렵게 있으며, 완성 된 게놈 년 Bgh 2의 L50 메가 12 6,989 supercontigs로 조립 하였다. 큰 게놈에도 불구하고, 흰가루병은 각각 년 Bgh 및 BGT 예측 5845 개 및 6540 유전자, 단백질 코딩 유전자의 수가 적은 것으로 보인다. 시퀀싱 흰가루병은 생존 11, 12, 13, 14 그들의 숙주 식물에 진균의 의존성과 일치 다른 진균류에 필수적인 최소한 99 핵심 유전자 결여 보인다. 텔로미어, 중심절 리보솜 RN 근처 반복 서열유전자 배열과 전이 될 풍부한 영역이 제대로 짧은 판독 시퀀싱 전략 조립 및 언더 표현 게놈 어셈블리 (15)에 종종있다. 이러한 영역은 게놈 서열에서 발생하는 공극의 많은 책임을 생각하고, 이것이 반복 요소 (16)의 그 광범위한 팽창으로 흰가루병에 적용된다. 높은 플라스틱 게놈 영역은 종종 반복적 인 지역 3에서 발견된다. 이들은 염색체 재 배열 사이트로서 기능하고 종종 이펙터 단백질을 코딩하는 유전자 및 이차 대사 효소를 코딩하는 유전자와 같은 강력한 선택 압력하에 유전자를 인코딩한다. 단일 분자 긴 읽기 시퀀싱 기술의 발전은 유전체 (15)의 반복적 인 지역에 걸쳐 순서에 대한 잠재적 인 솔루션을 제공합니다. 예를 들어, Faino 등. (2015)을 포함하여 긴 시퀀스 기술과 광 m 읽어 발견apping 유전자 주석 유전자 주석의 수를 증가 (및 일부의 수를 감소 시키거나 누락 게놈의 반복 DNA 서열의 비율 배로 그들을 곰팡이 병원균 Verticillium 달리아 두 균주 갭리스 게놈 시퀀스를 생성하도록 허용 ) 및 계시 게놈 17 재구성. 최소 크기&gt; 20 KBP 이러한 긴 읽기 시퀀싱 기술, 고품질의 게놈 DNA의 높은 농도를 사용하려면 필요하다. 여기에서 우리는 분생 포자로부터 포자 수집, 고 분자량의 DNA의 정제를위한 우리의 방법을 설명하고 흰가루병 종 Golovinomyces cichoracearum를 사용하여 우리의 품질 관리 평가는 오이 (18)에 성장. 이 프로토콜은 켈러 B. 실험 그룹 (쮜리히 대학, 스위스 취리히) (13), (19)에서 개발 된 프로토콜에 기초증가 DNA 수율 주도 여러 변형 및 크기의 DNA&gt; 48.5 KBP의 높은 비율을 포함한다. 이 프로토콜은 또한 연구소 20, 21, 22 에너지 공동 게놈의 미국학과의 추천에 따라 품질 관리 단계를 포함한다. 용해 완충액에 포함 된 각 시약 및 각 정제 단계에 근거하는 기능 등 (2008) 23 헨리에 기재되어있다. 식물과 미생물 조직에서 고 분자량 게놈 DNA의 분리에 사용할 수 게시 된 프로토콜은 또한이 프로토콜 24, 25, 26, 27, 28의 설계시 협의했다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MM400 Ball Mill</td>
			<td>Retsch</td>
			<td>20.745.0001</td>
			<td>Or equivalent ball mill</td>
		</tr>
		<tr>
			<td>2mL tubes for ball milling</td>
			<td>Sarstedt&#160;</td>
			<td>72.694.007</td>
			<td></td>
		</tr>
		<tr>
			<td>5/32&#34; stainless steel milling balls</td>
			<td>OPS Diagnostics</td>
			<td>GBSS 165-5000-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprocessor controlled 280 Series Water Bath Preset to 65&#176;C&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>2825</td>
			<td>Or equivalent water bath</td>
		</tr>
		<tr>
			<td>Microprocessor controlled 280 Series Water Bath Preset to 37&#176;C</td>
			<td>Thermo Fisher Scientific</td>
			<td>2825</td>
			<td>Or equivalent water bath</td>
		</tr>
		<tr>
			<td>Eppendorf 5417R refrigerated microcentrifuge</td>
			<td>Krakeler Scientific&#160;</td>
			<td>38-022621807</td>
			<td>Or equivalent microcentrifuge</td>
		</tr>
		<tr>
			<td>Chlorform:IAA (24:1 v/v)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C0549</td>
			<td>Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood</td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:IAA (25:24:1 v/v)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15593031</td>
			<td>Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood</td>
		</tr>
		<tr>
			<td>100% Isopropanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34923</td>
			<td>Chill to -20&#176;C prior use</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase, Dnase-free (10mg/ml)</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium metabisulfite</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Lauryl Sacroinate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>L9150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Cetyltrimethyl ammonium bromide (CTAB)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H6269</td>
			<td></td>
		</tr>
		<tr>
			<td>Uvex by Honeywell Futura Goggles S345C, Uvextreme</td>
			<td>Staples</td>
			<td>423263</td>
			<td>Or equivalent eyewear</td>
		</tr>
		<tr>
			<td>High Five COBALT nitrile gloves, LARGE</td>
			<td>Neta Scientific</td>
			<td>HFG-N193</td>
			<td>Or equivalent gloves</td>
		</tr>
		<tr>
			<td>GelRed Nucleic Acid Gel Stain 10,000X in water</td>
			<td>Biotium</td>
			<td>41003</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide 10mg/ml</td>
			<td>Biotium</td>
			<td>40042</td>
			<td>Hazardous in case of skin contact. Wear nitrile gloves and protective eyewear</td>
		</tr>
		<tr>
			<td>&#160;Agarose Ultrapure Bioreagent</td>
			<td>VWR International LLC</td>
			<td>JT4063</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1kb Plus DNA Ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>FERSM1332</td>
			<td></td>
		</tr>
		<tr>
			<td>8-48kb CHEF DNA size standards</td>
			<td>Bio-Rad</td>
			<td>170-3707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippin Prep&#160;&#160;</td>
			<td>Life Technologies Corporation</td>
			<td>4472172</td>
			<td>Or equivalent pulse-field gel aparatus</td>
		</tr>
		<tr>
			<td>Whatman&#160;qualitative filter paper, Grade 1</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>WHA1001042</td>
			<td>Or equivalent growth chamber</td>
		</tr>
		<tr>
			<td>Percival&#160; Growth Chamber</td>
			<td>Percival</td>
			<td>AR66LXC9</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 8000 UV-Vis Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-8000-GL</td>
			<td>Or equivalent spectrophotometer</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>P11496</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE Buffer (Tris-acetate-EDTA) (50X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>B49</td>
			<td>Dilute 1/50 with water before use (to 1X)</td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>12090015&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Borate-EDTA, 10X Solution (Electrophoresis)</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP13334</td>
			<td>Dilute 1/10 with water before use (to 1X)</td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td></td>
			<td></td>
			<td>Liquid nitrogen (-196 &#176;C) is a freezing hazard. It will also expand rapidly upon warming and should not keep in a tightly closed container</td>
		</tr>
	</tbody>
</table>

purification of high molecular weight genomic dna from powdery mildew for long-read sequencing

분체 곰팡이 곰팡이는 경제적으로 중요한 곰팡이 식물 병원균의 그룹입니다. 상대적으로 작은 인해 잘 발달 된 유전자와 게놈 자원의 부족 부분에 분자 생물학 및 병원체의 유전에 대한 알려져있다. 이 생물의 게놈 시퀀싱 및 조립 엄청나게 어려운 만든 대형, 반복적 인 게놈을 가지고있다. 여기서는 하나의 흰가루병 종 Golovinomyces cichoracearum에서 고 분자량의 게놈 DNA의 수집, 추출, 정제 및 품질 관리에 대한 평가 방법을 설명한다. 기술 된 프로토콜은 최적화 된 페놀 / 클로로포름 게놈 DNA를 추출하여 포자의 기계적 파괴를 포함한다. 전형적인 수율은 150 mg을 분생 당 7 μg의 DNA에 있었다. 이 절차를 이용하여 분리 된 게놈 DNA는 긴 판독 순서 (즉,&gt; 48.5 KBP)에 적합하다. 품질 관리 대책이 게놈 DNA의 크기, 수율 및 순도를 보장하기 위해 또한이 방법에 기재된. 여기에 설명 된 품질의 게놈 DNA의 염기 서열은 조립 및 차례로 더 나은 이해로 이어질 것입니다 여러 흰가루병 유전체의 비교를이 농업 병원균의 제어를 개선합니다.

G.로부터 정제 된 게놈 DNA를 60ng의 대표적인 예는 겔 전기 영동을 사용하여 아가로 오스 겔상에서 실행 cichoracearum 및 펄스 필드 겔 전기 영동을 사용하는 각각도 1 및도 2에 나타낸다. 소폭 통과하고 품질 관리를 실패 패스 게놈 DNA 제제를 나타낸다. 완벽한 품질 관리를 통과 게놈 DNA의 준비는 긴 읽기 게놈 시퀀싱 방법을 ?...

Watch this Scientific Journal Video about Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing at JoVE.com

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

긴 읽기 시퀀싱에 대한 흰가루병에서 높은 분자량의 게놈의 DNA의 정제

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

purification-high-molecular-weight-genomic-dna-from-powdery-mildew

Research

JoVE Journal

Genetics

11.6K 조회수.  University of California Berkeley. Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

비디오: Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

환자 조직에 대한 메틸 결합 DNA 캡처 시퀀싱

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

연구

유전학

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

차세대 시퀀싱에 적합한 식물성 세포질 DNA 농축 방법의 최적화 및 비교 분석

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

네이티브 Chromatin Immunoprecipitation Nucleosome 밀도 분석을 위한 라이브러리를 시퀀싱의 세대

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: 높은 처리량 시퀀싱 기반 기술을 늦게 게놈의 복제 확인

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

강력한 DNA 고립과 표 본관 표본에 대 한 높은 처리량 시퀀싱 도서관 건축

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

드문 이벤트 감지 오류 수정 DNA와 RNA 시퀀싱을 사용 하 여

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

격리, 시퀀싱 및 인간 중간 엽 줄기 세포에서 세포 외 MicroRNAs 분석

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

나노 포어 시퀀싱을 사용 하 여 전 혈에서 mRNA의 시퀀싱

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

RNA 시퀀싱을 사용하는 원형 RNA 식별

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

인간 다능성 줄기 세포에서 CRISPR-CAS9를 사용하여 정의된 게놈 수정 생성

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...