这项工作是由美国国立卫生研究院（1 R01 GM095384-01）授予森林Rohwer支持。我们感谢震中，一个Illumina的公司提供早期进入瑞博零流行病学套件。我们感谢马克哈塔伊的超速离心管架的设计和生产。我们感谢安德烈亚斯·阿斯和本杰明·诺尔斯关键读数和手稿的讨论，劳伦保罗协助拍摄过程。

注：诱导痰标本采集按照加州机构审查委员会的大学（HRPP 081500）和圣地亚哥州立大学机构审查委员会（IRB SDSU＃2121），由美国加州圣地亚哥大学的研究协调员（UCSD ）成人CF诊所。 1.样品采集和预处理（30 min内预治疗样品采集后） <ol><li>前样本采集，标记4 15ml试管为：（ⅰ）病毒宏基因组，（ⅱ）微生物宏基因组，（ⅲ）metatranscriptome，以及（iv）额外痰。重复每个样本。加2ml为0.1mm的二氧化硅珠粒到标有“Metatranscriptome”的管中，接着6毫升异硫氰酸胍基的RNA裂解缓冲液（GITC-裂解缓冲液）。 </li><li>期间样品收集，用无菌生理盐水溶液（60毫升）作为嗽口通过口服微生物以减少污染。采集痰标本在后一个30分钟的时间段吸入4毫升7％的高渗盐水通过喷雾器。过程样品立即，如下所述。 </li><li>将样品稀释至为8ml的总体积。 <ol><li>通过称重空痰杯之前和样品采集后估计样本量。 </li><li>如果样品的体积小于8毫升的0.02微米过滤的1×PBS中加适量以产生至少8毫升总样品体积。 </li><li>立即均质化样品用3毫升注射器，直到没有可见的团块残留痰内</li><li>使用同一注射器，制定2毫升痰，并立即着手步骤1.4。 </li></ol></li><li>从保存痰标本总RNA <ol><li>注入从步骤1.3.42毫升痰样品放入含有硅珠和GITC-裂解缓冲液的“metatranscriptome”管。 </li><li>盖上盖子，密封管安全地用封口膜，以避免泄漏。 </li><li>以中等速度为10英里立即同质化痰液ñ。取决于可用的涡流混合器，水平放置的管，如果需要，用胶带固定。 </li><li>保持所述管，在4℃或在冰箱和运输到实验室，如果必要的。 </li></ol></li><li>使用相同的注射器，等分2毫升痰的各成标有“病毒宏基因组”和“微生物宏基因组”，并从痰杯剩余痰转移到标有“额外痰”管中的管中。 </li><li>如果需要存储所有管在4℃或冰盒和运输到实验室。 </li></ol> 2.生成病毒宏基因组<ol><li>缓冲区和解决方案的研制<ol><li>制备的50mM二硫苏糖醇（DTT）提前，并储存在4℃。这是稳定2周。 </li><li>准备盐水镁（SM）的缓冲液（250毫升）：1 M氯化钠，的10mM 硫酸镁 ，50mM的Tris-盐酸;调节pH至7.4。过滤消毒（0.02微米孔径大小），并在室温下储存。 </li&gt; <li>根据Dornase单位/毫克干重定义的活动我准备的DNase I酶100U /μL（在分子级水）从冻干牛胰腺DNA酶。 </li><li>制备10×DNA酶I缓冲液（50毫升）：100mM的MgCl 2的，20毫米氯化钙2;调节pH至6.5。过滤消毒（0.02微米孔径大小），并在室温下储存。 </li><li>制备4％的多聚甲醛。 </li><li>制备200倍的TE缓冲液：的2M的Tris-HCl（pH为8.5），0.2M EDTA中。过滤消毒（0.02微米孔径大小），并在室温下储存。 </li><li>准备使用分子级水加入10ml 10％十二烷基硫酸钠（SDS）。 </li><li>采用分子级水准备50毫升CTAB /氯化钠（10％CTAB 700毫米氯化钠）。溶解CTAB过夜。如果析出物仍然存在，加热该溶液在65℃。解决方法是在常温下的高粘度。 注意：在使用0.02微米的过滤器缓冲器的过滤使溶液中的除去的病毒样颗粒，但不游离核酸污染。 </li></ol></li><li>样品前处理<ol><li>准备新鲜的6.5二硫苏糖醇（DTT）适量。 </li><li>通过加入0.02微米过滤的SM缓冲液，以产生6毫升总体积稀释的匀浆。 </li><li>为了有助于黏液溶解，加入6.5毫摩尔二硫苏糖醇（DTT）到样品等体积（6毫升）中，涡流剧烈混合，并培育1小时，在37℃。 </li><li>涡旋处理过的样品，并剧烈旋转在10℃，3056×g离心15-20分钟。 </li><li>收集上清液到新的15ml试管中。 </li><li>重复步骤2.2.3和2.2.5的下一个样品。 </li><li>转移和过滤用0.45μm过滤器安装在注射器上清液到新的15ml试管中。 注：如果过滤器堵塞，检索从注射器样品和省略过滤步骤。 </li><li>取0.45微米过滤样品的100μl的子样本，进行氯仿和DNA酶I处理（见SEction 2.3.12-2.3.15）并加入4％多聚甲醛的量等于固定样品为荧光显微镜（ 图2A）。 </li><li>对于一个“包罗万象”的病毒颗粒富集方法（见讨论），转到步骤2.3.12省略病毒颗粒选择基于氯化铯梯度离心。然而，这可能会导致在氯仿抗性细菌污染并在病毒裂解物更高量的宿主的DNA。 </li></ol></li><li>病毒样颗粒（VLP）浓缩和纯化<ol><li>通过将氯化铯与未过滤的SM缓冲液适量至所需密度制备单独的氯化铯（氯化铯）溶液（1.7微克/毫升，1.5微克/毫升，1.35微克/毫升和1.2微克/毫升）。过滤通过使用前一个0.02微米的过滤器的每个解决方案。 </li><li>设置CsCl梯度， 如图2B所示 。 </li><li>负载1毫升1.7微克/毫升到每个管中，负载1毫升1.5克/毫升到每个管中，负载1毫升1.35克/毫升到每个管中，负载为1.2g / ml的成每个管（可选），最后装入6-8毫升样品到各自的管中。标记各个层来表示每个级分的位置。 </li><li>平衡各反对对管内1毫克。 </li><li>每个管小心装入旋转桶。所有旋转桶，即使它们是空的。加载自旋桶到转子。 </li><li>离心机在82844 xg离心在4℃下2小时。 </li><li>离心后，小心地取出从支架管而不破坏密度梯度。 </li><li>使用3毫升注射器用18G的针，刺穿管只是为1.5g / ml的密度层下方（ 图2，红色箭头）和拉〜1.5毫升入注射器。 </li><li>通过缓慢除去针，并允许在管中的剩余部分滴入到一个新的15ml试管通过穿刺收集上层级分。这种标签“上浪费分数”。 </li><li>收集1.5克/毫升fractioN（含有的VLP）从注射器通过喷射的内容到两个新的微量离心管中。 </li><li>所有样品重复步骤2.3.8-2.3.10。 </li><li>加入0.2体积的氯仿至病毒浓缩物，剧烈振荡，在室温下孵育10分钟，自旋以最大速度进行5分钟，并收集水相。 </li><li>加10倍的DNase缓冲液和DNA酶I（终浓度= 2.5单位/微升）到氯仿处理的病毒浓缩液，孵育在37℃下为1.5-2小时。 </li><li>灭活在65℃下的DNA酶活性15分钟。 </li><li>除去15微升氯仿和DNA酶I处理过的病毒级分的进新管中，并加入15微升4％多聚甲醛固定的样品进行荧光显微镜。 </li></ol></li><li> DNA提取<ol><li>池的病毒浓缩物从每个样品倒入清洁蒸压50毫升橡树岭高速离心管中。 </li><li>添加以下：0.1体积200倍的TE缓冲液，10微升的0.5M EDTA的每毫升第充足，1卷甲酰胺，和10微升糖原。搅拌均匀，室温孵育30分钟。 </li><li>使用新的卷，添加2倍体积室温的100％乙醇。拌匀，孵育在4℃至少30分钟。 </li><li>粒料的DNA通过使用的SS-34转子纺纱管在17226×g离心20分钟，在4℃下。 </li><li>通过使用血清吸管小心弃去上清液。用冰冷的70％乙醇两次清洗沉淀。 </li><li>除去尽可能多的液体尽可能并允许沉淀风干，在室温下搅拌15分钟。 </li><li>重悬在567微升1×TE缓冲液（pH8.0）中的DNA沉淀。 注：允许至少15分钟，在室温下完全再悬浮。过夜储存的DNA再悬浮在4℃直至进一步处理。 </li><li>传送整个567微升重新悬浮的DNA溶液到新的1.5 ml离心管。添加30微升预温热的10％SDS和3μl的蛋白酶K（20 _6;克/毫升），调匀，并培育1小时，在56℃。预暖的CTAB / NaCl的在65℃。 </li><li>加入100微升5 M氯化钠调匀。添加预热的CTAB / NaCl溶液，涡流80微升，并孵育10分钟，在65℃。 </li><li>添加氯仿，涡旋混合，并旋等体积在16100×g离心5分钟。 </li><li>上清转移到一个新的1.5 ml离心管。添加苯酚/氯仿，涡旋混合，并旋等体积的在16100×g离心5分钟。 </li><li>上清转移到一个新的1.5 ml离心管。添加氯仿，涡旋混合，并旋等体积在16100×g离心5分钟。 </li><li>上清转移到一个新的1.5 ml离心管。添加异丙醇等体积的上清液部分，混合，并孵育在-20℃至少30分钟。 </li><li>沉淀DNA，旋在16100 XG为15分钟，在4℃。移液器关闭上清小心地用冰冷的70％乙醇洗涤沉淀两次。 </l我&gt; <li>执行短的自旋，并从管除去剩余的乙醇。空气干燥沉淀15分钟。 </li><li>重悬DNA沉淀用50μl洗脱缓冲液（5毫摩尔Tris，pH值8.5）。允许沉淀再水合至少5分钟，在室温下进行。 </li><li>使用高灵敏度的基于荧光的测定法量化的DNA。 </li></ol></li><li>扩增使用Phi29聚合酶（可选） <ol><li>制备2×退火缓冲液：80mM的Tris-盐酸（pH8.0）中，20毫的MgCl 2。 </li><li>淡化Phi29 DNA聚合酶5U /μL。 </li><li>预混样品缓冲液，由50微升的随机六聚体引物（100μM），125微升2×退火缓冲液，和25微升水中。分装和储存在-20℃。 </li><li>预混反应缓冲液，由100微升Phi29 10x缓冲液，40微升10mM的dNTPs和560微升水。分装和储存在-20℃。 </li><li>加入1μl模板DNA到4微升样品缓冲液。 </li><li>孵化日Ë混合物在95℃下进行3分钟，并在冰上冷却。 </li><li>从2.4.4添加14微升反应缓冲液到混合物中，通过上下吹打混匀。 </li><li>加入1μlPhi29 DNA聚合酶，通过上下吹吸混合，孵育在30℃下进行18小时，随后65℃10分钟。 </li><li>清理使用基因组DNA柱或酚/氯仿和乙醇沉淀反应。 </li></ol></li><li>荧光显微镜（参考哈斯等人。2014 28过滤系统设置） 注意：下面的分离和纯化，荧光显微镜用核酸染料可用于验证病毒颗粒的样品中的存在和纯度（ 图2A和2C）。样品中游离DNA可以产生高背景荧光。因此，该样品应当DNA酶I处理过的前固定和染色的显微照片。 <ol><li>制备安装溶液（0.1％抗坏血酸，50％甘油）中。加入100微升10％抗坏血酸4.9 1X磷酸盐缓冲液（PBS）的毫升，调匀。加入5 ml 100％甘油的混合物中，充分混合并标记管作为“装载”。 </li><li>滤波器安装用0.02微米的氧化铝基质的一次性注射器过滤，等分入微量离心管，并储存在-20℃。 </li><li>等份加入100μl样品到一个新的离心管，并加入4％多聚甲醛的量等于固定的VLP。在室温下孵育该混合物至少10分钟。 </li><li>补足体积至1ml，加入800微升0.02微米过滤的水。加入1微升的SYBR金染色到管并且在室温下孵育10分钟。 </li><li>通过转动-9和-10磅（-62.1和-68.9千帕）之间的真空泵上设置的过滤系统。 </li><li>用水冲洗所述基座和放置一个0.02微米的氧化铝基质滤波器环形聚丙烯支撑环到过滤器底座。 </li><li>放置一个过滤器塔在过滤器底座与所述过滤器和安全用夹子的顶部。 </li><li>从吸取1.5 ml离心管中的内容进入过滤塔，并允许几分钟样品过滤通过。 </li><li>标签和吸管10微升安装试剂到微观的幻灯片。 </li><li>离开同时去除过滤塔和夹子上的真空。 </li><li>小心地取出从过滤器底座的过滤器和吸干滤波器用Kimwipe的底部，然后直接放置在过滤器上的安装的顶部上的显微镜载玻片。 </li><li>吸取另外10微升安装试剂到过滤器，并放置盖玻片上的过滤器。 </li></ol></li></ol> 3.生成微生物宏基因组<ol><li>缓冲液和溶液的制备<ol><li>准备加入50ml 1×DNA酶缓冲液：50mM醋酸钠，10mM的MgCl 2的，2mM的氯化钙 ;调节pH至6.5。过滤消毒（0.22微米）和存储在室温下。 </li><li>根据Dornase单位/毫克干重定义的活动我准备的DNase I酶1000U /μL（在分子级水）从冻干牛胰腺DNA酶。 </li><li>制备100ml的SE缓冲液：75mM氯化钠，25mM的EDTA;调节pH至7.5。过滤消毒（0.22微米），并储存在室温下。 </li></ol></li><li>样品前处理之前，提取DNA <ol><li>通过加入5倍体积的0.22微米过滤的1×PBS稀释的匀浆。例如，加入10 mL 1X PBS的分成2毫升的样品。 </li><li>添加β巯基乙醇至2％（V / V）的最终浓度。岩石在室温下将该混合物（在化学罩）2小时。 </li><li>旋转的样品在10℃和3056×g离心15分钟，并弃去上清液。 </li><li>重悬沉淀在10毫升的分子级水（或0.22微米的过滤后的水），并在室温下孵育15分钟。 </li><li>重复步骤3.2.3和3.2.4一次。 </li><li> SPIÑ​​在10℃和3056×g离心15分钟，并弃去上清液。 </li><li>悬浮颗粒在5毫升1X DNA酶缓冲和每毫升样品中加入15微升的DNase I（1000 U /微升）。 </li><li>孵育在37℃下反复混合2小时。 </li><li>灭活在65℃下的DNA酶活性15分钟。 </li><li>旋在10℃和3056×g离心15分钟，并弃去上清液。重悬沉淀在10ml SE缓冲器。 </li><li>重复步骤3.2.10。 </li><li>旋在10℃和3056×g离心15分钟，并弃去上清液。 </li><li>重悬沉淀在2ml SE缓冲液，并转移到2微量离心管中。 </li><li>沉淀在离心管的细胞。旋管在16100×g离心，在室温下搅拌15分钟。 </li><li>除去上清液，然后从沉淀的细胞用基因组DNA提取试剂盒，革兰氏阳性变异协议中提取的DNA。 </li></ol></li></ol> 4.生成Metatranscriptome <ol><li>样品预- 处理<ol><li>样品收集和均化后，立即执行细胞中GITC-裂解缓冲液珠击的机械裂解。参见步骤1.4。 </li><li>旋转，在4℃下，将混合物600×g离心5分钟以沉淀二氧化硅珠粒。 </li><li>转移上清液至新管。 </li><li>加入200μl的氯仿，每750微升GITC-裂解缓冲液使用时，用手剧烈摇动15秒，在室温下孵育10分钟，并旋在4℃和3056×g离心15分钟。在这15分钟的旋转，准备步4.2。 </li><li>在15分钟的自旋（含水相相间有机相形成一个明确的分离）后，提取水相（而不破坏相间）到新的无RNase管（多个）。 注：该水相含有的RNA。保持在冰上的管中，直到下一个步骤。 </li></ol></li><li>使用可商购的基于列的RNA纯化试剂盒或便利着想执行总RNA的提取和纯化ntional异丙醇为基础的RNA沉淀。 <ol><li>硅胶柱为基础的RNA纯化<ol><li>测量得到的水性级分的总体积。 </li><li>添加的RNA结合的缓冲区适当的音量来样拌匀。 </li><li>调节混合物根据制造商的协议到适当的结合状态。拌匀，做短的自旋。 </li><li>加载混合物进入RNA的列。对于大体积的样品，使用多个加载和加载每列高达4倍。否则，考虑使用多列每个样品。 </li><li>根据制造商的方案适当地洗柱。 </li><li>洗脱RNA与至少30微升不含RNA酶的水中。双洗脱将略微增加RNA的产率。然而，这会冲淡RNA浓度。 </li><li>测量RNA浓度，直接进行DNA酶I处理。使用生物分析仪来检查RNA的质量（推荐）。 </li></oL&gt; </li><li> RNA降水<ol><li>添加异丙醇等体积的（ 例如 ，500微升异丙醇入500微升水相）和2微升的10微克/微升不含RNA酶的糖原至样品。 </li><li>在室温下孵育该混合物10分钟。 </li><li>旋在12,000 xg离心4℃15分钟。 </li><li>小心取出上清液，加入1 ml无RNA酶的75％乙醇。在7500×g离心，4℃5分钟旋转的混合物，以确保颗粒是完整的。 </li><li>小心地取出乙醇。 </li><li>重复步骤4.2.2.4 4.2.2.5和一次。 </li><li>空气干燥沉淀10分钟。 </li><li>水合于50μl不含RNA酶的水沉淀，孵育在55℃进行5分钟，并直接进行DNA酶处理。使用生物分析仪来检查RNA的质量（推荐）。 </li><li>商店RNA的等分试样在-20℃，或-80℃下长期贮存。 </li></ol></li></ol></li></oL&gt;

The authors have nothing to disclose.

病毒宏基因组学病毒颗粒是使用聚乙二醇（PEG）沉淀或小体积浓缩浓缩。在某些情况下，浓度可能并不需要，但预过滤或低速离心步骤用于除去真核生物和微生物细胞。病毒裂解液将进一步丰富和使用密度梯度离心9,41或小尺寸的过滤器（ 例如 ，0.45微米），以去除真核和大微生物细胞25。纯化典型地密集，但惰性溶液如蔗糖或氯化铯以分离和浓缩的病毒颗粒41的密度梯度超离心。物理分离是基于大小和病毒颗粒的浮力密度。因此，过滤器的孔径适当选择和梯度的严谨制备是必不可少的分离特定的病毒群体，作为体力恢复的成功的VLP确定社会隔绝41（即不通过提取密度内的过滤器或下降，即 ，病毒颗粒不会在宏基因组被检测）。后的病毒分离和浓度，可能有污染的非病毒基因组物质同时存在于游离核酸​​和微生物和真核细胞的形式在样品中。因此，关键是要验证的病毒颗粒在样品的纯度（ 图1A和1B）。甲氯仿处理通常用于裂解剩余的细胞，随后通过核酸酶处理以降解游离核酸之前的核酸提取。 一个警告，以所呈现的工作流程是使用密度梯度分离，分离病毒颗粒，因为它可能会排除包膜病毒颗粒，可能是太浮力进入CsCl梯度。另一种“包罗万象”的方法是忽略密度梯度组合通道ATION和分离从0.45微米的社区的DNA - 用氯仿和DNA酶I.这种方法处理过的滤液也适合容纳小体积的样品，如那些从拭子或血浆。然而，这可能会导致在氯仿抗性的细菌污染和较高量的DNA酶I的抗胞外DNA。 目前测序协议要求的核酸测序文库制备1毫微克至1微克即较高的DNA产量提供了测序的选择更广泛的选择。产生viromes的DNA浓度往往范围低于检测极限，以超过200毫微克/微升。回收的病毒核酸的量可能不足以直接测序文库的制备。在这种情况下，核酸扩增是必需的。接头扩增鸟枪文库（LASLs）2,42,43和基于多重置换扩增（MDA）的全基因组扩增是两个方法最常用的，以产生足够的DNA测序。丙二醛的方法，例如那些基于Phi29 DNA聚合酶是已知的，从扩增偏倚之苦，并且可以优先地扩增的ssDNA和环状DNA，产生非定量分类学和功能表征44,45。所述LASLs方法的优化版本已被证明引入只有很少的偏见，促进更高的灵敏度（对于少量的起始材料），并容易适于不同测序平台43。然而，该方法具有许多步骤，需要专门的设备，以减少DNA的损失，并且限制为双链DNA模板。在我们的实验室中，这种方法已被成功地适应于扩增的DNA，从支气管lavage-，coral-和海水衍生的VLP（未发表和Hurwitz 等。46）中提取的可检测和不可检测量。 开发数据分析管道具有CLAssically一直是病毒性宏基因组分析中最具挑战性的一个方面，由于病毒社区的高度多样化和鲜为人知的性质。虽然估计有10 8病毒基因型生物圈，迄今为止当前病毒数据库包含〜4000病毒基因组，这是该近似的总病毒多样性的约1 / 100,000。因此，基于相似性检索（如BLAST 47）的分类和功能分配在病毒宏基因组具有内在的挑战。许多序列不具有显著相似之处在数据库中的基因组，并且因此，被分类为不明。尽管同源性为基础的搜索是用于分配分类学中最重要的应用和功能的序列数据，已经开发了基于数据库独立分析的替代方法48- 50。 Fancello 等 51提供的维拉使用的计算工具和算法进行全面审查升宏基因组学。 微生物宏基因组学通常情况下，DNA的低渗裂解处理的微生物群落（HL-DNA）萃取的总量范围为20纳克至5微克。的产率是高度依赖于病人的健康状况和痰样品的采集量，这解释见于的HL-DNA在这项研究中（ 表2）中提取的总产率的变化。的关键步骤，以产生良好的质量的序列数据依靠产生测序文库的质量。 图2显示了典型的尺寸范围为使用酶基的DNA片段化过程从CF痰衍生的微生物的DNA中产生的测序文库。最优文库大小是依赖于测序平台和应用程序的选择，并且因此，分片程序可以优化，如果需要的话，通过各种途径，如超声处理和雾化。此外所提出的有代表性的结果，所提出的方法对CF痰从在多个时间点的多个患者收集的成功也示于Lim 等人（2012）9和Lim 等人。（2014）10。 以往的研究表明9,10每个病人怀有一套独特微生物群落，从而改变随着时间的推移，从而反映了社区内的主要参与者的持久性，而波动可能是由于扰动，如抗生素治疗。这些波动无论是每天发生，即使没有外部干扰或由于抽样程序和来样加工，仍然是一个问题。基于所述的HL-DNA的宏基因组和16S rDNA的扩增子的分析上，在7天的纵向取样表明微生物型材无抗生素扰动的日常波动（第1天，2和3）中的最小的（ 图3A和3B）。经介绍口服抗生素duction马上第3天取样后，改变了社区概况成为第4天明显虽然抗生素环丙沙星针对已知的细菌性病原体如铜绿广谱假单胞菌 ， 金黄色葡萄球菌 ， 链球菌和肺炎 ，治疗增加P的相对丰度假单胞菌同时减少链球菌属 。和P.黑素 。 6日，社会慢慢恢复到初始起点群落结构。结果表明，单一患者内的微生物型材波动更可能是由于在气道社区扰动。 定的16S rDNA的库和宏基因组从HL-来源的DNA的微生物轮廓之间的一致性，我们排除了从本研究中使用的16S rRNA基因的引物起始的偏见。一个可能的解释看出整个16S rDNA序列分类的差异从总DNA和HL-衍生的DNA（ 图3B）产生人型材可以是大量的假单孢菌属的存在。抗生素治疗后细胞外的DNA。这是支持的结果，这些差异是在第7天最为明显，在抗生素治疗，其靶向假单胞菌后三天。除了其他。环丙沙星是常用的第一线治疗的CF患者和慢性P.绿脓杆菌感染，尽管其活动范围包括了大部分CF相关的病原体。我们假设，抗生素治疗根除易感社区包括链球菌属 。因此创建填充性P.利基绿脓杆菌 。 绿脓杆菌可能获得抵抗通过增加其生物膜群落和细胞外的DNA已被证明是其生物膜结构52的主要结构支撑。甚至为t他群落结构恢复，外DNA可能留在CF痰。因此，这些数据表明，低渗裂解和呈现在这个工作流程中，不仅除去来自人的DNA的潜在受益的洗涤步骤，但也微生物衍生的胞外DNA，可能歪曲实际微生物轮廓。 Metatranscriptomics 高品质的metatranscriptome应包含相对较少的核糖体RNA（rRNA基因）序列和代表社会成绩单（mRNA）的一个公正的采样。由于半衰期短和有限数量的mRNA，这是至关重要的协议，如这里介绍的，最大程度地减少样品处理，以最大限度地提高转录回收的数量。 在最近几年，已经开发出并适于在可商购的试剂盒几种方法为rRNA的枯竭。这些措施包括为MicroB 充实瑞博零和样本的具体消减ħybridizations 53是基于寡核苷酸杂交，并与mRNA-ONLY试剂盒是基于含有5&#39;单磷酸外切核酸酶活性的靶向的RNA。此外，也可几种方法表达富集，如MessageAmp II细菌试剂盒的优先polyadenylates和放大线性RNA。其中的一些方法（ 例如，表达-ONLY，为MicroBË随心而MessageAmp）同时使用达到最佳效率。然而，所有这些方法的效力是有限的，以部分降解的rRNA加工特别是当，如经常在从CF样本中提取总RNA观察到。聚腺苷酸化依赖性RNA扩增，不能用于生成metatranscriptomes由两个真核和原核mRNA的。另外，该聚（A）尾加到序列可以减少的有用的序列数据的量。与均聚物延伸区域将倾向于有质量得分较低，Causing一个显著数目的读取要被过滤掉通过测序和后序软件，平均有用读取长度修剪掉聚后（A）尾将被显著54减小。 与复杂的CF微生物群落和部分降解RNA（ 图4A和4C）处理，我们以前的研究表明，由瑞博零黄金套件杂交捕获方法更有效地去除人类和微生物的rRNA基因相比，使用相结合的治疗其它试剂盒9（ 表3）。所得到的数据使人类宿主和微生物成绩单并发分析。取决于产量和RNA的质量，以及测序平台的最终选择，许多这些工艺包括cDNA合成步骤可以简化与测序文库生成。例如，核糖零处理的RNA可用于使metatranscriptome测序天秤里斯使用ScriptSeq RNA测序文库制备试剂盒。 动物相关社区的宏基因组分析提供了包括主机和与其相关的社区整体功能实体的一个综合表述。这里提出的工作流程是适应于各种复杂的动物相关联的样本，特别是那些含有稠粘液，高量的细胞碎片，细胞外的DNA，蛋白质和糖蛋白复合物，以及除宿主细胞至所需的病毒和微生物颗粒。即使病毒和微生物颗粒可以在每一步会丢失，颗粒的分离和纯化是必要的，以减少主机的DNA的量。而宏基因组学数据提供了研究社区的代谢潜力，metatranscriptomics通过揭示编码功能9的差异表达补充这一点。基因组学和成绩单的数据进行了全面评估，取得了新的插件ights社区相互作用的动力学和促进改善的疗法9,10,55的发展。

<html>与人体相关联的病毒和微生物群落已被广泛地研究，在过去十年中，通过测序技术1,2中的应用。该成果已使人们认识到的重要性微生物在人类健康和疾病。主要的倡议来自人类微生物项目描述的细菌（和一些古细菌）驻留在人的皮肤，并且在口服空腔，呼吸道，泌尿生殖道，和胃肠道3。通过支气管肺泡灌洗（BAL）4,5和鼻咽拭子4健康人的气道的微生物进一步的研究表明，肺可以作为环境取样装置中，导致短暂的微生物定植在呼吸道。然而，微生物定植在受损气道表面的影响，可能导致严重的和慢性肺部感染，如那些见于囊性纤维化（CF）的患者。 吨“&gt; CF为引起的囊性纤维化跨膜调节因子（CFTR）的基因突变6一个致命的遗传性疾病。这些突变引起有缺陷的CFTR蛋白，这反过来影响跨越上皮的顶端表面跨上皮离子迁移的疾病影响多个器官系统，但大多数发病率和死亡率的可归因于CF肺病7时CF肺提供用于微生物建群的唯一的生态系统。在离子迁移的缺陷会导致粘液在CF气道建立，产生的微环境，包括有氧，由静态营养丰富的粘膜表面锚定微需氧和厌氧室中。这有利于环境和定殖微生物的增殖，包括病毒，细菌和真菌。急性和慢性肺部微生物感染导致恒定的，但是效果不佳的免疫反应，从而导致广泛的气道重塑，肺容量损失，最终低O2高CO2进制失败。 与CF肺相关的细菌群落都使用这两种文化的依赖和文化独立的方法，其中包括使用16S核糖体RNA（rRNA）的基因测序8和猎枪宏基因组9,10得到了很好的描述。在16S rRNA基因为基础的方法能够刻画各种各样的微生物物种和捕捉群落多样性广泛的转变。然而，在其第界定社区限制（总结于CLAESSON 等，2010 11）和代谢潜力的预测被限制为已知为确定的分类单元的那些一般功能。因此，16S rRNA基因测序的方法是不够的存在于CF肺部的多种微生物群落的必要分类和功能的分析的精度。这里介绍的宏基因组方法的补充16S rRNA基因为基础的方法，克服了它的局限性，并实现了相对有效的办法分析这两种微生物群落的分类和CF肺部遗传内容。 微生物的DNA来自动物相关样品中分离往往含有大量的宿主DNA。 CF痰或肺组织样品通常含有大量的人类DNA由嗜中性粒细胞在免疫反应释放时，经常是大于99％的总DNA 12 - 14。虽然一些完整的人类细胞可以存在，最该DNA的是游离在溶液中或吸附到微生物的表面上。此外，特别粘稠粘液插头，细胞碎片和其它未知杂质的存在下进一步复杂化的微生物细胞的分离。几种方法进行了测试消耗这些样品的人类DNA，其中包括的Percoll梯度从微生物细胞15分开的人力，用DNA酶I处理，溴化乙锭单氮化物，以选择性地降解人类DNA 16和MolYsis试剂盒，都用有限的成功。<html>迄今最有效的微生物的DNA纯化步骤的CF痰液已经由布莱滕等人所描述的方法的变形例。（1995）17。这种方法，在此被称为低渗裂解（HL）方法，使用β巯基乙醇的组合，以减少粘蛋白二硫键，真核细胞的低渗裂解和DNA酶I处理的DNA溶于9。尽管缺乏替代品的HL方法提出由于（1）有些担忧，从微生物不必要的裂解和可能导致的偏见（ii）在群落组成9,10观察到的波动是否与样品加工相关的变化的神器。除了猎枪宏基因组的产生，我们通过比较总DNA和微生物的DNA来自使用同一组来自整个连续七天单个患者采集的痰样品的HL法提取的16S rRNA基因型材解决这些问题。 ENT“&gt;相较于微生物群落，病毒性社区与动物相关的特征是有限的18,19在CF呼吸道病毒社区只得到最低限度的特点20 - 22第一个宏基因组研究中的病毒特征社区在CF气道的DNA表明，与CF肺部相关大多数病毒是噬菌体20。噬菌体在CF和非CF个体的代谢潜力显著不同，具体地，在CF个体噬菌体群体进行基因反射细菌宿主的适应与CF气道的生理学，和在CF肺组织的病毒细菌毒力20，后续的宏基因组的研究表明解剖区域22之间的病毒群落明显的空间异质性，此外，CF肺组织窝藏最低的病毒多样性观察迄今在任何生态系统22。鉴定大多数病毒是噬菌体与潜在感染的CF病原体。然而，也发现真核病毒如疱疹病毒，腺病毒，和人乳头状瘤病毒（HPV）。在一个事件中，其中，解剖过程中观察到的肺组织囊肿，超过99％的人类乳头状瘤病毒基因组的回收，即使病人从未诊断患有肺乳头状瘤或癌。这表明病毒多样性本不仅反映组织损伤的严重程度，但也可能暴露和解释一个潜在未表征的疾病。这里描述的方案提供了一种简单而功能强大的方式来隔离从样本包括大量厚的粘液，宿主和微生物细胞，游离的DNA，以及细胞碎片的病毒样颗粒（VLPs）。 宏基因组学的补充，metatranscriptomics用于整个微生物群落与宿主9,23监测基因表达的动态。在这种情况下，无论是微生物和何吨的mRNA需要被优先选择。因为细菌的mRNA聚腺苷酸化不是，一个寡-dT系的mRNA下拉方法不能被利用。聚腺苷酸化依赖性RNA扩增不能主机相关联的样本中被使用，如果将样品已知含有大量的真核mRNA的。许多动物相关的样本，包括CF痰，含有细胞的除了高含量的细胞碎片和核酸包括RNA酶的高密度。因此，另一种具有挑战性的任务是防止metatranscriptome处理期间广泛RNA降解。在大多数情况下，从CF痰提取总RNA是部分降解，限制了来自RNA的下游应用和效用。在最近几年，已经开发出并适于在可商购的试剂盒几种方法为rRNA的枯竭。这些方法的功效但是有限的，尤其是与部分降解的rRNA 9,24工作时。这里所采用的方法允许编为适于高效下游总的rRNA去除部分降解的总RNA的检索。从比较两种不同的试剂盒部分降解的总RNA中rRNA的去除效率的直接比较由Lim 等说明。（2012）9。 总体而言，本手稿的目的是提供一套完整的协议（ 图1），以产生病毒和微生物猎枪宏基因组，以及一个metatranscriptome，从单一动物相关的样本，利用诱导痰样本作为一个例子。分子实验室工作流程应包括独立前和扩增后的区域，以减少交叉污染。该方法是很容易适应其他类型的样品例如组织22，鼻咽和口咽拭子25，支气管肺泡灌洗（BAL）和珊瑚（未发表数据）。每个样品应立即收集后进行处理尤其是当微生物宏基因组，并会见atranscriptomics研究是期望的。如果将样品冷冻，它限制了完整的微生物细胞的分离微生物宏基因组作为冷冻潜在破坏细胞的完整性。然而，冷冻并不排除metatranscriptomics和病毒分离，但RNA的回收可通过冻融过程受到影响病毒颗粒的质量和数量。要注意的是诱导痰一直作为样品与成人CF患者和其他慢性肺部疾病26,27相关联的BAL可太侵入性的许多研究的主要来源是重要的。在我们的研究中，痰样本，收集与一个仔细和一致的抽样方法， 例如 ，下面的漱口水，并使用无菌生理盐水溶液，以保持口腔微生物污染的痰样品到最低限度内口腔冲洗。

<table border="0" cellpadding="0" cellspacing="0" width="741">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Guanidine isothiocyanate-based RNA lysis buffer</td>
			<td style="width:119px;">Life Technologies</td>
			<td>10296-028</td>
			<td>TRIzol LS Reagent was used in this study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silica beads; 0.1 to 0.15 mm</td>
			<td style="width:119px;">Cole-Parmer</td>
			<td>YO-36270-62</td>
			<td>Zirconia-Silicate Beads were used in this study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol Molecular Grade (Dry Powder)</td>
			<td style="width:119px;">Promega</td>
			<td style="width:119px;">V3155</td>
			<td>Can be purchased from any other company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile syringe filter; 0.45 &#181;m pore size hydrophilic PVDF membrane</td>
			<td style="width:119px;">Millipore</td>
			<td style="width:119px;">SLHV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I enzyme&#160;</td>
			<td>Calbiochem</td>
			<td align="right">260913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Sterile syringe filter; 0.02 &#181;m pore size</td>
			<td style="width:119px;">FisherScientific</td>
			<td style="width:119px;">09-926-13</td>
			<td>Diameter: 25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Ultra-Clear Centrifuge Tubes</td>
			<td style="width:119px;">Beckman</td>
			<td align="right">344059</td>
			<td>9/16 X 3 &#189; in. (14X90mm), suitable for SW41 Ti Rotor and VLPs density gradient ultracentrifugation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW41 Ti Rotor</td>
			<td style="width:119px;">Beckman</td>
			<td align="right" style="width:119px;">333790</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:337px;">SS-34 Fixed Angle Rotor</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">28020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Phi29 DNA polymerase</td>
			<td>Monserate Biotech</td>
			<td align="right" style="width:119px;">4001</td>
			<td>10 U/&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Phi29 Random Hexamer</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">S0181</td>
			<td>Previously bought from Fidelity Systems</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alumina matrix filter with annular polypropylene support ring; 0.02 &#181;m pore size</td>
			<td style="width:119px;">FisherScientific</td>
			<td style="width:119px;">09-926-34</td>
			<td>Whatman Anodisc Filter Membranes were used in this study; Diameter 25mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:337px;">SYBR Gold Nucleic Acid Gel Stain</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">S-11494</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">2-Mercaptoethanol</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:119px;">M6250-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free DNase I&#160;</td>
			<td style="width:119px;">NEB</td>
			<td style="width:119px;">M0303S</td>
			<td>Can be purchased from any other company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Glycogen, RNA grade</td>
			<td style="width:119px;">FisherScientific</td>
			<td style="width:119px;">FERR0551</td>
			<td>Can be purchased from any other company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Oak Ridge high-speed centrifuge tubes</td>
			<td style="width:119px;">FisherScientific</td>
			<td style="width:119px;">05-562-16B</td>
			<td>For large volume DNA extraction procedure, suitable for SS-34 fixed-angle rotor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Total rRNA removal kit</td>
			<td style="width:119px;">Epicentre</td>
			<td style="width:119px;">MRZE724</td>
			<td>ScriptSeq Complete Gold Kit (Epidemiology) can be used to couple rRNA removal with sequencing library preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Cesium chloride&#160;</td>
			<td style="width:119px;">FisherScientific</td>
			<td style="width:119px;">BP1595-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Hexadecytrimethyl-ammonium bromide (CTAB)</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:119px;">H5882-100G</td>
			<td></td>
		</tr>
	</tbody>
</table>

purifying the impure: sequencing metagenomes and metatranscriptomes from complex animal-associated samples

高通量测序的访问已经彻底改变了生物学的许多领域。为了更好地了解主机相关病毒和微生物群落，全面的工作流用于DNA和RNA提取被开发了。工作流同时生成病毒和微生物宏基因组，以及metatranscriptomes，从下一代测序单个样品。这些方法的耦合提供两者的分类学特性和社区编码功能的概述。所提出的方法使用囊性纤维化（CF）的痰，一个有问题的样本类型，因为它是非常粘稠的，并含有高量的粘蛋白，中性粒细胞游离的DNA，和其他未知的污染物。这里所描述的协议针对这些问题，并成功地恢复病毒和微生物DNA以最少的人类DNA污染。为了配合宏基因组学研究中，metatranscriptomics协议进行了优化，以恢复这两种微生物和主机的mRNA包含相对少的核糖体RNA（rRNA）的序列。的数据特性的概述呈现给作为评估的方法成功的基准。额外的CF痰液样品也收集到（ⅰ）评估跨越连续七天的微生物分布的一致性单个患者中，和（ii）比较的宏基因组的方法的一致性为16S核糖体RNA基因为基础的测序。结果表明，微生物型材无抗生素扰动每日波动​​是最小的，并在共同的CF相关的细菌的分类学概况分别来自低渗裂解（HL）衍生的DNA所产生的16S rDNA的库和宏基因组之间高度相似。但是，从总DNA和HL-来源的DNA产生16S rDNA的分类学轮廓之间的差异表明，低渗裂解和洗涤步骤中，不仅除去来自人的DNA的受益，而且微生物衍生extracellulAR DNA可能会歪曲实际的微生物的分布。

Watch this Scientific Journal Video about Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples at JoVE.com

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

使用囊性纤维化气道为例，手稿呈现全面的工作流包括宏基因组和metatranscriptomic方法的组合来表征动物相关样品中的微生物和病毒的社区。

从复杂的动物相关样品测序宏基因组和Metatranscriptomes：净化不纯

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

purifying-impure-sequencing-metagenomes-metatranscriptomes-from

Research

JoVE Journal

Biology

37.1K 次观看.  San Diego State University. 使用囊性纤维化气道为例，手稿呈现全面的工作流包括宏基因组和metatranscriptomic方法的组合来表征动物相关样品中的微生物和病毒的社区。 

视频: Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

科研

生物学

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

肚子里的龙虾总夹层， Homarus Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the internal status of the cell. Glycans on tumor cells tend to have abundant sialic acid and fucose moieties. We propose that these cancer-associated glycan variants be exploited for biomarker development aimed at diagnosing early-stage disease. Accordingly, we developed a mass spectrometry-based workflow that incorporates chromatography on affinity matrices formed from lectins, proteins that bind specific glycan structures. The lectins Sambucus nigra (SNA) and Aleuria aurantia (AAL), which bind sialic acid and fucose, respectively, were covalently coupled to POROS beads (Applied Biosystems) and packed into PEEK columns for high pressure liquid chromatography (HPLC). Briefly, plasma was depleted of the fourteen most abundant proteins using a multiple affinity removal system (MARS-14; Agilent). Depleted plasma was trypsin-digested and separated into flow-through and bound fractions by SNA or AAL HPLC. The fractions were treated with PNGaseF to remove N-linked glycans, and analyzed by LC-MS/MS on a QStar Elite. Data were analyzed using Mascot software. The experimental design included positive controls&mdash;fucosylated and sialylated human lactoferrin glycopeptides&mdash;and negative controls&mdash;high mannose glycopeptides from Saccharomyces cerevisiae&mdash;that were used to monitor the specificity of lectin capture. Key features of this workflow include the reproducibility derived from the HPLC format, the positive identification of the captured and PNGaseF-treated glycopeptides from their deamidated Asn-Xxx-Ser/Thr motifs, and quality assessment using glycoprotein standards. Protocol optimization also included determining the appropriate ratio of starting material to column capacity, identifying the most efficient capture and elution buffers, and monitoring the PNGaseF-treatment to ensure full deglycosylation. Future directions include using this workflow to perform mass spectrometry-based discovery experiments on plasma from breast cancer patients and control individuals.

Lectin-conjugated POROS beads were employed for HPLC. Glycopeptide standards served as positive and negative controls. MARS-14 depleted, trypsin-digested human plasma was chromatographed and flow-through (FT) and bound fractions collected for ESI-LC-MS/MS analyses. Glycopeptides were enriched in the bound fraction as compared to FT.

一个外源凝集素的高效液相色谱法从复杂生物样品，以丰富的选择性糖基化肽

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

定量组织样本中的γH2AX灶

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

从巨 Unilamellar 泡中提取膜纳米管

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

食品和环境样品中沙门氏菌的准总体基因体分析

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.