我们感谢万实验室和Nagarajan实验室的成员进行了详细的讨论。 N.Nagarajan得到A * STAR的资助。姚先生获得了A * STAR和Science-Branco Weiss奖学金社会的资助。

1.用生物素化补骨脂素和RNA提取处理HeLa细胞<ol><li>在10cm板中补充有10％胎牛血清（FBS）和1％青霉素链霉素（PS）的Dulbecco改良的Eagle培养基（DMEM）中培养HeLa细胞。 </li><li>用5毫升1x PBS洗涤HeLa细胞两次。通过将盘垂直放置1分钟，将多余的PBS完全从盘中排出。 </li><li>向细胞中均匀加入含有200μM生物素化补骨脂素和0.01％w / v卵磷脂的1mL PBS，并在37℃下孵育5分钟。虽然生物素化的补骨脂素可以进入细胞，当细胞暴露于少量的洋甙素（0.01％）5分钟时，其渗透性要高得多</li><li>取出含有处理过的HeLa细胞的10厘米平板的盖子，将盘子平放在冰上。使用UV交联剂，将含有细胞的培养皿用365nm紫外线照射20分钟，距离UV灯泡3厘米的距离。 </li><li>隔离t根据制造商的方案，通过硫氰酸胍 - 苯酚 - 氯仿提取来自HeLa细胞的RNA。加入1体积的异丙醇以在室温下沉淀RNA 30分钟。 暂停点:RNA可以无限期地在-20°C储存在异丙醇中。可以在该步骤中进行斑点印迹以检查生物素标记的补骨脂素是否已经成功进入细胞并且在体内具有交联的RNA（ 图2 ）。 </li><li>在4℃下以20,000×g离心沉淀的RNA 30分钟以回收RNA。去除上清液，并加入1 mL 70％冷乙醇至RNA颗粒中以洗涤RNA。 </li><li>在4℃下将样品以20,000×g离心15分钟。取出上清液，并在室温下空气干燥沉淀5分钟。 </li><li>向RNA沉淀中加入无核酸酶的水，并使用紫外光谱仪定量RNA的浓度。 暂停点:f。可在-80°C储存RNA睫毛在液氮中冻结。 </li></ol> RNA分裂<ol><li>在0.2 mL PCR管中预热15μL2x RNA片段化缓冲液和10μLRNA样品（1μg/μL），95°C 1 min。通过上下移液将10μL加热的2X RNA片段缓冲液与10μLRNA样品混合。在95℃孵育样品5分钟。通过在冰上冷却反应停止反应。 </li><li>将2个碎裂反应转移并汇集到1.5 mL离心管中。向反应液中加入40μL无核酸酶的水，10μL3 M乙酸钠，1μL糖原和300μL100％乙醇进行裂解反应。将RNA在-20℃孵育至少1小时以沉淀RNA。 </li><li>将沉淀的RNA以20,000×g离心30分钟，除去上清液并用1mL 70％乙醇洗涤RNA。 </li><li>将RNA以20,000 xg离心15分钟，上清，并将RNA溶解在20μL无核酸酶的水中。 </li></ol> RNA大小选择和洗脱<ol><li>向微量离心管中回收的RNA中加入20μLRNA加载染料。用RNA加载染料在95℃加热RNA 3分钟，并将其置于冰上2分钟。 </li><li>在微量离心管中加入3μLRNA加载染料至0.25μL10 nt DNA梯。将梯子在95℃下加热3分钟，并将其放在冰上2分钟。 </li><li>将变性的梯子和样品加载到8.6厘米x 6.8厘米6厘米的TBE尿素凝胶上，并在180℃下通过电泳电泳40分钟。将凝胶在10mL含有1:10,000核酸凝胶染色剂的TBE缓冲液中染色5分钟在黑暗中。 </li><li>使用24 G针刺穿底部的干净的0.6 mL微量离心管，并将其置于2 mL微量离心管中。 </li><li>使用透照器观察后染色凝胶上的条带，并切割对应于90-11的凝胶切片0点将凝胶切片转移到2 mL微量离心管中穿刺的0.6 mL微量离心管中。执行该大小选择以允许嵌合片段具有最小长度以映射到转录组，并在下游尺寸选择步骤中区分接近连接的产物与未产生的产物。 </li><li>将凝胶切片在室温下以12,000xg离心2分钟，以切碎凝胶切片并将其收集在2mL微量离心管中。丢弃空的0.6 mL离心管。向2 mL微量离心管中的切碎的凝胶切片中加入700μL洗脱缓冲液，并在4℃下恒温孵育过夜，以使样品扩散到缓冲液中。 </li><li>将凝胶切片和洗脱缓冲液转移到离心管过滤器中，并以20,000 xg离心2分钟。凝胶片将被捕获在过滤器的顶部隔间。丢弃凝胶片和顶部隔间。 </li><li>加入1μL糖原，7将100μL异丙醇加入到微量离心管中的滤液中，并将RNA在-20℃下沉淀至少1小时或过夜。 暂停点:RNA可以无限期地在-20℃下在异丙醇中沉淀。 </li><li>在4℃下以20,000×g离心沉淀的RNA 30分钟以回收RNA。取出上清液，加入1 mL 70％冷乙醇洗涤RNA沉淀。在4℃下以20,000xg离心洗涤的沉淀15分钟。 </li><li>取出洗涤缓冲液，加入20μL无核酸酶的水重新悬浮RNA。使用UV光谱仪定量RNA的浓度。 暂停点:RNA可以在-80°C储存，在液氮中快速冷冻。 </li></ol> 4. RNA交联区域的丰富<ol><li>涡旋链霉抗生物素蛋白包被的磁珠强力地将细珠重新悬浮在管中。将100μL珠子转移到1.5 mL离心管中，并将其放在磁铁上ic放置1分钟以使珠子粘附到管的磁性侧。小心地从小珠中取出储存缓冲液，并将管从磁铁架上取出。 </li><li>向微量离心管中的珠子中加入1 mL裂解缓冲液，上下移液。将含有微珠的微量离心机放置在磁性架上1分钟，以将珠子与洗涤缓冲液分离。重复这一次总共3次洗涤。 </li><li>通过将含有微珠的微量离心机放置在磁性架上1分钟，将洗涤缓冲液从珠粒中取出。将RNA酶抑制剂（1:200）加入裂解缓冲液中，并将100μL裂解缓冲液加入到微量离心管中洗涤的珠粒上。 </li><li>加入2mL新鲜制备的杂交缓冲液，1mL补充的裂解缓冲液，1.5μg大小分级的RNA和100​​μL重悬珠珠至15mL锥形离心管中。轻轻旋转管。 </li><li>将15 mL锥形离心管在37°C下孵育30分钟，端对端旋转。在37°C预洗洗涤缓冲液。 </li><li>将15 mL锥形离心管放在磁性支架上15 mL管中2 min，将珠子与缓冲液分离。将缓冲液从管中小心取出并将其丢弃。 </li><li>将1mL预热的洗涤缓冲液加入到珠中，上下移液并将珠转移到1.5mL的微量离心管中。在37℃下，在珠子上孵育5分钟，进行端对端旋转。 </li><li>简单地离心微量离心管的内容物。将微量离心管中的1.5 mL洗涤珠置于磁性支架上2 mL试管中1分钟，以将珠子与洗涤缓冲液分离。从微量离心管中轻轻移取缓冲液，从珠中取出缓冲液。 </li><li>将1mL预热的洗涤缓冲液加入到珠子中并向上和向下移液以重复洗涤。共执行5次洗涤。在第五次洗涤结束时，通过将含有微珠的微量离心机放置在磁体架上来除去洗涤缓冲液1分钟。 </li></ol>邻近连接<ol><li>将1 mL冷T4多核苷酸激酶（PNK）缓冲液加入到洗涤的珠中，并在4℃下孵育珠子5分钟，并以端对端旋转。将含有珠粒的微量离心管置于磁条上1分钟，轻轻取出T4 PNK缓冲液。重复此步骤总共2次洗涤，并在最后一次洗涤后取出T4 PNK缓冲液。 </li><li>根据表1将缓冲液加入珠中。为了使RNA片段的3&#39;末端能够连接到下面的3&#39;衔接子上，将缓冲液中的珠子在37℃下恒温搅拌4小时，将RNA末端的3&#39;环状磷酸酯基团转化为3&#39;OH。 </li><li>根据表2添加缓冲液到前面的反应。为了使RNA片段的5&#39;末端具有连接能力，在37℃下在ATP存在下恒定搅拌下孵育反应1小时将RNA上的5&#39;OH转化为5&#39;磷酸。 </li><li>根据表3 ，将缓冲液加入前一反应进行接近连接。在16℃下反应16h恒温搅拌， </li><li>将含有连接的RNA的微量离心机置于磁体架上1分钟。小心取出上清液，并加入1 mL室温洗涤缓冲液至珠粒。通过上下移动重悬。 </li><li>将微量离心管放置在磁体架上1分钟，并小心地取出洗涤缓冲液。执行总共2次洗涤，并在第二次洗涤结束时从珠中除去洗涤缓冲液。 </li><li>加入100μL的RNA PK缓冲液加入珠中，并通过上下移液重悬。将样品在95℃加热10分钟。 </li><li>将样品在冰上冷却1分钟，并向样品中加入500μL硫氰酸胍 - 苯酚 - 氯仿。通过剧烈涡旋混合10秒。孵化mixtu在室温下放置10分钟。 暂停点:RNA可以在-80°C下储存在硫氰酸胍 - 苯酚 - 氯仿中几个月。 </li><li>向硫氰酸胍 - 苯酚 - 氯仿提取样品中加入100μL氯仿。大力涡旋10秒。在4℃下将样品以20,000×g离心15分钟。 </li><li>将400μL水层转移到新的1.5 mL微量离心管中。加入800μL（2体积）100％乙醇，并通过上下移液混合。 </li><li>将RNA溶液转移到RNA清除柱上，按照制造商的说明回收RNA，确保即使保留了小RNA。在100μL无核酸酶的水中洗脱RNA。 暂停点:RNA可以在-80°C储存，在液氮中快速冷冻。 </li></ol> 6.生物素化补骨脂素的反向交联<ol><li>将100μL洗脱的样品转移到24孔板的孔中吃了。取下盖子并在UV 254nm下照射样品5分钟。 </li><li>将反向交联的样品转移到干净的微量离心管中。加入10μL乙酸钠，1μL糖原和300μL100％乙醇，并将RNA在-20℃下沉淀至少1小时或过夜。 暂停点:RNA可以无限期地在-20℃下在乙醇中沉淀。 </li><li>在4℃下以20,000×g离心沉淀的RNA 30分钟以回收RNA。取出上清液，加入1 mL 70％冷乙醇洗涤RNA沉淀。 </li><li>在4℃下以20,000xg离心洗涤的沉淀15分钟。取出洗涤缓冲液，加入4.25μL无核酸酶的水重新悬浮RNA。将RNA转移到0.2 mL PCR管中。 暂停点:RNA可以在-80°C储存，在液氮中快速冷冻。 </li></ol>反转录和cDNA循环<ol><li>加入0.75μL的RNA接头（0.5μg/μL）重悬于PCR管中，并在80℃加热90秒。将样品放在冰上2分钟。 </li><li>根据表4 ，将缓冲液添加到3&#39;接头连接的变性样品中。在25℃下孵育反应2.5小时。 </li><li>将反应转移到1.5 mL离心管中。向反应液中加入90μL无核酸酶的水，然后加入10μL乙酸钠，1μL糖原和300μL100％乙醇。将RNA在-20℃沉淀至少1小时或过夜。 暂停点:RNA可以无限期地在-20℃下在乙醇中沉淀。 </li><li>在4℃下以20,000×g离心沉淀的RNA 30分钟以回收RNA。取出上清液，加入1 mL 70％冷乙醇洗涤RNA沉淀。 </li><li>在4℃下以20,000g离心洗涤的沉淀15分钟。取出洗涤缓冲液，将沉淀物重新悬浮在5μL无核酸酶的水溶液中亚特。 </li><li>向微量离心管中回收的RNA中加入5μLRNA加载染料。用RNA加载染料在95℃加热RNA 3分钟，并将其置于冰上2分钟。 </li><li>在微量离心管中加入3μLRNA加载染料至0.25μL10 nt DNA梯。将梯子在95℃下加热3分钟，并将其放在冰上2分钟。 </li><li>使用8.6 cm x 6.8 cm 6％TBE尿素凝胶进行尺寸分级，如步骤3.3和3.4所示。 </li><li>使用透照器显现用核酸染色的凝胶，并在梯子上切下对应于110-140nt的凝胶切片。将凝胶切片转移到2 mL微量离心管中穿刺的0.6 mL微量离心管中，并按照步骤3.6 - 3.9的方案进行。 </li><li>加入5μL无核酸酶的水重悬RNA颗粒。将RNA转移到0.2 mL PCR管中。 暂停点:RNA可以在-80°C储存，在液氮中快速冷冻。 </li><li>在1.25加入1μLRT引物81; M至PCR管中的RNA，并在80℃加热2分钟。将样品放在冰上2分钟。 </li><li>根据表5添加反转录缓冲液。在50℃下反应30分钟。 </li><li>向反应中加入1.1μL1N NaOH，在98℃加热20分钟。将反应放在冰上。 </li><li>将反应转移到1.5 mL离心管中。向反应液中加入90μL无核酸酶的水，然后加入10μL乙酸钠，1μL糖原和300μL100％乙醇。将DNA在-20℃沉淀至少1小时或过夜。 暂停点:DNA可以无限期地在-20℃下在乙醇中沉淀。 </li><li>在4℃下将沉淀的DNA以20,000g离心30分钟以回收DNA。取出上清液，加入1 mL 70％冷乙醇洗涤DNA沉淀。在4℃下以20,000xg离心洗涤的沉淀15分钟。取出洗涤缓冲液重悬放入5μL无核酸酶的水。 </li><li>使用8.6 cm x 6.8 cm 6％TBE尿素凝胶进行尺寸分级，如步骤3.3和3.4所示。 </li><li>使用透照器显现后染色凝胶，并在梯子上切下对应于200-240nt的凝胶切片。由于加入96碱基RT引物，逆转录的cDNA现在比RNA片段长96个碱基。将凝胶切片转移到2 mL微量离心管中穿刺的0.6 mL微量离心管中。 </li><li>将凝胶切片在室温下以12,000xg离心2分钟，以切碎凝胶切片并将其收集在2mL微量离心管中。丢弃空的0.6 mL离心管。 </li><li>加入700μL洗脱缓冲液至2 mL离心管中切碎的凝胶切片，37°C恒温恒温孵育2 h。按照步骤3.7-3.8中所述的协议。 </li><li>加入6μL无核酸酶的水重新悬浮DNA沉淀。将DNA转移到0.2 mL PCR管中。 <li>将缓冲液加入到用于cDNA环化的反应中，根据表6 。将反应在60℃下孵育1小时，并在80℃下加热10分钟以使反应失活。 </li><li>将反应转移到1.5 mL离心管中。根据制造商的说明，使用DNA清除柱纯化环化的cDNA。向柱中加入20μL无核酸酶的水洗脱纯化的cDNA。 暂停点:DNA可以在-20°C储存几个月。 </li></ol> 8. PCR扩增（小规模PCR） <ol><li>将缓冲液加入根据表7的反应进行PCR扩增，以测试文库扩增所需的最小循环次数。 </li><li>根据表8设置PCR循环条件。暂停反应并转移5μL将PCR反应在10,15和20个循环中分离成分离的1.5mL离心管。 </li><li> 在不同周期的5μLPCR反应中加入1μL6x DNA凝胶加载染料。在3V琼脂糖凝胶电泳上以120V进行凝胶电泳1小时以显现PCR产物。 <ol><li>使用最低数量的PCR周期（Y），其显示在约250个碱基处的扩增（ 图3中 ，在15个循环的扩增周期和10个周期的微弱条带中存在强带），12个大规模PCR扩增循环可能生成足够的深度测序材料，因为使用的cDNA量是小规模PCR扩增的10倍）。 </li></ol></li></ol> 9. PCR扩增（大规模PCR）和纯化<ol><li>将缓冲液添加到根据表9的反应中进行大规模PCR的PCR扩增。 </li><li>根据PCR设置PCR循环条件表10 。 </li><li>将5μL6x DNA凝胶加载染料加入到25μLPCR反应中，并加载到3％琼脂糖凝胶的孔中。将1 kb加DNA梯子加入一个单独的孔。在100 V下进行凝胶电泳1.5 h。 </li><li>使用UV透射仪可视化完成的凝胶。 </li><li>提取含200-300个碱基的PCR产物的凝胶切片，并将凝胶转移到干净的2mL离心管中。 </li><li>从DNA凝胶提取试剂盒中加入1 mL凝胶溶解度缓冲液至凝胶切片，并在室温下孵育30分钟，或直到凝胶溶解，不断搅拌。 </li><li>向溶解的凝胶中加入200μL异丙醇，并通过移液进行良好的混合。将700μL样品转移到DNA凝胶提取清洗柱中，以13,000 xg离心30秒。继续将溶解的样品转移到色谱柱中，直到所有样品都装载到色谱柱上。 </li><li>加入500μL凝胶溶解度缓冲液，然后将700μL柱洗涤缓冲液加入柱中，根据制造商的方案。将12μL无核酸酶的水加入到色谱柱的中心以排除DNA。暂停点:DNA可以保存在-20°C。 </li><li>使用荧光定量法定量DNA的浓度。如果构建多个库并将其汇总在一起进行测序，则将相同量的每个样品添加到池中，以进行高通量测序。 </li></ol>

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Y. <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+mapping+of+RNA+structure+using+nuclease+digestion+and+high-throughput+sequencing.">Genome-wide mapping of RNA structure using nuclease digestion and high-throughput sequencing.</a> Nat.Protoc. 8 (5), 849-869 (2013).</li><li>Wan, Y., 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=Landscape+and+variation+of+RNA+secondary+structure+across+the+human+transcriptome.">Landscape and variation of RNA secondary structure across the human transcriptome.</a> Nature. 505 (7485), 706-709 (2014).</li><li>Spitale, R. 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作者没有竞争的经济利益。

在这里，我们详细描述了SPLASH的实验和计算工作流程，这是一种允许我们以全基因组方式识别成对RNA相互作用的方法。我们已经在细菌，酵母和人类文化中成功地利用SPLASH，并且预计该策略可以广泛应用于不同细胞状态下的多种生物。协议中的关键步骤之一是从至少20μg的交联RNA开始，为下游过程提供足够的材料。然后将RNA片段化至100个碱基并进行PAGE大小选择。这些步骤对于我们在图书馆生成过程中优先富集连接的嵌合体而不是单体是重要的。我们通常在碎裂和第一次大小选择后恢复约1.5μg的RNA，然后根据SPLASH工作流程将RNA转化为cDNA文库。我们发现这一数量的起始RNA使我们能够为h生成足够的材料使用少于15个循环的PCR扩增的高通量测序。随着PCR循环次数的增加，PCR复制事件的数量急剧增加，将扩增循环数量保持在较低水平是至关重要的，以便能够在下游分析中提取有用的和独特的嵌合体。 与其他基于全基因组补骨脂素的策略相反，SPLASH利用生物素化版本的补骨脂素将碱基配对的RNA片段彼此交联。当我们将交联量相对于每一百五十个碱基进行大约一个交联时，我们可以通过在RNA片段化后使用链霉抗生物素蛋白珠来富集交联的RNA区域。此外，当RNA结合在珠上时进行酶反应也使我们能够方便地进行缓冲液交换和洗涤。然而，策略的局限性之一是生物素化的补骨脂素在补骨脂素或4&#39;-氨基甲基三oxsalen（AMT）。因此，确保生物素化补骨脂素进入细胞并有效交联RNA是至关重要的。我们常规地将交联的提取的RNA与生物素化的寡核苷酸一起作为阳性对照进行点印迹，以确保我们的RNA正确交联。在交联弱的情况下，可以使用渗透细胞膜的策略，例如加入低浓度的黄体素（至0.01％）5分钟，以允许生物素化的补骨脂素有效进入细胞。由于补骨脂素以与核酸（UV 260nm）相同的波长吸收，我们通常使用荧光定量系统，而不是UV吸收来定量交联的RNA。 基于补骨脂素的交联策略的一个限制是补骨脂素优先在尿苷（U）处交联。因此，在交联过程中可能会错过U型的碱基配对区域。因此，同时检测交联事件b两条链提供了相互作用发生的证据，缺乏交联并不等同于缺乏相互作用。在我们的SPLASH协议中，我们捕获了很少的microRNA-mRNA相互作用，并且少量的lncRNA相互作用。由于microRNA结合的mRNA可能在基因表达中下调，并且lncRNA通常低表达，它们在我们的数据中的差的表达主要是由于测序深度不足。我们通常为每个重复序列每个人转录组文库至少2亿个配对末端读数。在这个深度上，我们的大部分测序读取都是相当丰富的RNA。我们预计，针对特定RNA群体的浓缩策略的使用将大大增强这些相对较低丰度RNA的信号。我们在分析嵌合数据中观察到的另一个挑战是区分真正的相互作用嵌合事件与拼接产生的嵌合体。防止污染of拼接读取我们的嵌合体列表，我们过滤掉与转录组中已知注释剪接位点相近的所有读数。我们预计，通过更完整的拼接注释，嵌套的最终列表将变得更加准确。 与以前针对单个RNA物种或单一RNA结合蛋白特异性的RNA相互作用的策略不同，SPLASH以所有RNA的全基因组方式绘制RNA-RNA相互作用的能力使我们能够研究大规模RNA互动网络，并首次发现新的分子内和分子间RNA相互作用。使用SPLASH，我们在人和酵母转录组中获得了数千个分子内和分子间的相互作用，使我们能够在体内瞥见RNA相互作用的组织和动力学。将这种长距离RNA相互作用数据整合到RNA结构建模算法中的进展很有可能改进我们目前的RNA组织体内模型。将SPLASH纳入分子间RNA预测算法，如snoRNA预测程序，也可以提高这些预测的准确性。我们预计未来SPLASH在其他复杂生物和动态系统中的应用将继续揭示基于RNA的基因调控在生物学中的复杂性。

研究大分子如何折叠和相互作用是理解细胞中基因调控的关键。在过去十年中，为了了解DNA和蛋白质如何促进基因调控，在过去十年中已经做了很多努力，对基因表达的转录后调控相对较少。 RNA在其线性序列及其二级和三级结构中携带信息1 。与其自身和其他物质配对的能力对于其在体内的功能是重要的。高通量RNA二级结构探测的最新进展已经为转录组2,3,4,5,6,7,8中的双链和单链区域的位置提供了宝贵的见解关于配对互动伙伴关系的信息仍然很大程度上缺失。为了确定哪个RNA序列与转录组中的另一个RNA区域相互作用，我们需要全球成对的信息。 以全球，公正的方式映射成对的RNA相互作用传统上是一个重大的挑战。虽然以前的方法，如CLASH 9 ，hiCLIP 10和RAP 11被用来以大规模的方式鉴定RNA相互作用，但是这些技术通常将RNA碱基配对映射到与特定蛋白质或RNA物种相互作用的RNA亚类。研究全球RNA相互作用的最新进展包括方法RPL 12 ，其不能在体内稳定RNA相互作用，因此可能仅捕获体内相互作用的子集。为了克服这些挑战，我们和其他人开发了全基因组，无偏见的策略p RNA 在体内相互作用，使用修饰版本的交联剂补骨脂素13,14,15 。在本协议中，我们描述了使用生物素化的补骨脂素在体内交联碱基配对RNA，然后进行邻近连接和高通量测序的P soralen交联，Ligigated和S选择Hyrbrids（SPLASH）的S等位基因的细节。识别RNA基因配对伙伴全基因组（ 图1 ） 15 。 在本手稿中，我们描述了使用培养的贴壁细胞进行SPLASH的步骤，在这种情况下是HeLa细胞。相同的方案可以容易地适应于悬浮哺乳动物细胞和酵母和细菌细胞。简言之，HeLa细胞用生物素化的补骨脂素处理并在365nm下照射以在体内交联相互作用的RNA碱基对。然后从细胞中提取RNA，用链霉抗生物素蛋白珠片段碎片化并富集交联区域。然后使用接近连接将相互作用的RNA片段连接在一起，并制成用于深度测序的cDNA文库。测序后，将嵌合RNA映射到转录组/基因组上以鉴定彼此配对的RNA相互作用区域。我们已经成功地利用SPLASH在酵母和不同人类细胞中识别数千个RNA相互作用，包括分子内和分子间RNA碱基配对在不同类别的RNA，如snoRNA，lncRNA和mRNAs，以瞥见结构组织和相互作用模式的细胞中的RNA。

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		<col />
		<col span="3" />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">1 Kb Plus DNA Ladder</td>
			<td style="width:284px;">Life Technologies Holdings Pte Ltd</td>
			<td style="width:284px;">10787026</td>
			<td style="width:284px;">DNA ladder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 bp DNA ladder</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>10821-015</td>
			<td>DNA ladder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20 % SDS solution</td>
			<td>First BASE</td>
			<td>BUF-2052-1L&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20x SSC</td>
			<td>First BASE</td>
			<td>BUF-3050-20X1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3.0 M Sodium Acetate Solution</td>
			<td>First BASE</td>
			<td>BUF-1151-1L-pH5.2&#160;&#160;</td>
			<td>Required for nucleic acid precipitation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40% Acrylamide/Bis Solution, 19:1</td>
			<td>Bio-Rad</td>
			<td>1610145</td>
			<td>TBE Urea gel component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ambion Buffer Kit</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>AM9010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium Persulfate, Molecular Grade&#160;&#160;</td>
			<td>Promega</td>
			<td>V3131&#160;</td>
			<td>TBE Urea gel component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bromophenol Blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloroform</td>
			<td>Merck</td>
			<td>1.02445.1000</td>
			<td>RNA extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Single strand DNA ligase</td>
			<td>Epicentre</td>
			<td>CL9025K</td>
			<td>CircLigase II ssDNA Ligase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tube filters</td>
			<td>Sigma-Aldrich</td>
			<td>CLS8160-96EA</td>
			<td>Costar Spin-X centrifuge tube filters</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">D5628-1G DIGITONIN CRYSTALLINE</td>
			<td>Sigma-Aldrich</td>
			<td>D5628-1G</td>
			<td>For cell treatment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dark Reader Transilluminator&#160;</td>
			<td>Clare Chemical Research</td>
			<td>Dark Reader DR89X Transilluminator</td>
			<td>Blue light transilluminator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Gel Loading Dye (6X)</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>R0611</td>
			<td>Required for agarose gel electroporation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Pan BioTech</td>
			<td>P04-03500</td>
			<td>For Hela cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin magnetic beads</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>65002</td>
			<td>Dynabeads MyOne Streptavidin C1&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stand for 15ml tubes</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>12301D</td>
			<td>DynaMag-15</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnetic stand for 15ml tubes</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>&#160;12321D</td>
			<td>DynaMag-2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ThermoMixer</td>
			<td>Eppendorf</td>
			<td>5382 000.015</td>
			<td>Eppendorf ThermoMixer&#160;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Biotinylated psoralen</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>29986</td>
			<td>EZ-Link Psoralen-PEG3-Biotin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">F8T5/BL&#160;</td>
			<td>Hitachi</td>
			<td>F8T5/BL&#160;</td>
			<td>365 nm UV bulb</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>10270106&#160;&#160;</td>
			<td>Components of Hela medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formamide</td>
			<td>Promega</td>
			<td>H5052&#160;&#160;</td>
			<td>Component in hybridization buffer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">G8T5</td>
			<td>Sankyo-Denki</td>
			<td>G8T5</td>
			<td>254 nm UV bulb</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycogen&#160;</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>10814010</td>
			<td>Required for nucleic acid precipitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanodrop</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>Nanodrop 2000</td>
			<td>Spectrophotometer for nucleic acidquantification</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease free water&#160;</td>
			<td>First BASE</td>
			<td>BUF-1180-500ml&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin Streptomycin&#160;&#160;</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>15140122&#160;&#160;</td>
			<td>Components of Hela medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-Fidelity DNA polymerase (2x)</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>F531L&#160;</td>
			<td>Phusion High-Fidelity PCR Master Mix with HF Buffer (2x)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primers Set 1</td>
			<td>New England Biolabs</td>
			<td>E7335L&#160;</td>
			<td>PCR primers</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA Gel Extraction Kit</td>
			<td>QIAgen</td>
			<td>28106</td>
			<td>QIAquick Gel Extraction Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorometric Quantification kit</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>Q32854</td>
			<td>Qubit dsDNA HS Assay Kit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA clean up kit</td>
			<td>Qiagen</td>
			<td>74106</td>
			<td>RNeasy Mini Kit Silica-membrane column</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rnase Inhibitor</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>AM2696</td>
			<td>SUPERase In</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reverse transcriptase</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>18080400</td>
			<td>SuperScript III First-Strand Synthesis SuperMix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nucleic Acid Gel Stain</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>S11494</td>
			<td>SYBR GOLD NUCLEIC ACID 500 UL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201L</td>
			<td>End repair enzyme</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 RNA Ligase 1</td>
			<td>New England Biolabs</td>
			<td>M0204L</td>
			<td>Enzyme for proximity ligation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 RNA Ligase 2, truncated KQ</td>
			<td>New England Biolabs</td>
			<td>M0373L</td>
			<td>Enzyme for adaptor ligation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Temed</td>
			<td>Bio-Rad</td>
			<td>1610801</td>
			<td>TBE Urea gel component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Guanidinium thiocyanate-phenol-chloroform</td>
			<td>Life Technologies Holdings Pte Ltd</td>
			<td>15596018</td>
			<td>TRIzol&#174; Reagent for RNA extraction</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Urea</td>
			<td>First BASE</td>
			<td>BIO-2070-5kg&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV crosslinker</td>
			<td>Stratagene</td>
			<td>400072</td>
			<td>UV Stratalinker 1800</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV Transilluminator</td>
			<td>UVP</td>
			<td>95-0417-01</td>
			<td>For visualizing of bands</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylene Cyanol FF</td>
			<td>Sigma-Aldrich</td>
			<td>X4126-10G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA cleanup it</td>
			<td>Zymo Research&#160;</td>
			<td>D4004</td>
			<td>Zymo DNA concentrator-5&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">List of software required</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FastQC software</td>
			<td>0.11.4</td>
			<td>http://www.bioinformatics.babraham.ac.uk/projects/fastqc/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SeqPrep software</td>
			<td>1.0.7</td>
			<td>https://github.com/jstjohn/SeqPrep</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BWA software</td>
			<td>0.7.12</td>
			<td>http://bio-bwa.sourceforge.net/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SAMTOOLS software</td>
			<td>1.2.1</td>
			<td>http://www.htslib.org/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PULLSEQ software</td>
			<td>1.0.2</td>
			<td>https://github.com/bcthomas/pullseq</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">STAR software</td>
			<td>2.5.0c</td>
			<td>https://github.com/alexdobin/STAR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rem_dups.py PYTHON script</td>
			<td>PYTHON 2.7.11</td>
			<td>https://github.com/CSB5/splash/tree/master/src</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">find_chimeras.py PYTHON script</td>
			<td>PYTHON 2.7.11</td>
			<td>https://github.com/CSB5/splash/tree/master/src</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pickJunctionReads.awk AWK script</td>
			<td>AWK GNU 4.1.2</td>
			<td>https://github.com/CSB5/splash/tree/master/src</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buffer composition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elution buffer:&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.3 M sodium acetate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysis Buffer:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mM Tris-Cl pH 7.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mM EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1% SDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Always add Superase-in fresh before use except when washing beads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K Buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mM NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10 mM TrisCl pH 7.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mM EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% SDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hybridization Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">750 mM NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1% SDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 mM Tris-Cl pH 7.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mM EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15% formamide (store in the dark at 4 &#176;C)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Always add Superase-in fresh before use</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x SSC Wash Buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2x NaCl and Sodium citrate (SSC) (diluted from 20x SSC Invitrogen stock)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% SDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X RNA fragmentation buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">18mM MgCl2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">450mM KCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">300mM Tris-CL pH 8.3</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x RNA loading Dye:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">95% Formamide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.02% SDS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.02% bromophenol blue</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.01% Xylene Cyanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1mM EDTA&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3&#39; RNA adapter sequence</td>
			<td>5rAppCTGTAGGCACCATCAAT/3ddC</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RT primer sequence</td>
			<td>AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC/iSp18/CACTCA/iSp18/TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

mapping rna-rna interactions globally using biotinylated psoralen

了解RNA与自身和其他人的相互作用是了解细胞中基于RNA的基因调控的关键。虽然已经显示RNA-RNA相互作用（例如微小RNA-mRNA相互作用）的实例调节基因表达，但是在细胞中发生RNA相互作用的完全程度仍然是未知的。以前研究RNA相互作用的方法主要集中在与特定蛋白质或RNA物种相互作用的RNA亚类。在这里，我们详细介绍了一种名为S等位基因的交联S型， L型化和S型选择性蛋白（SPLASH）的方法，其允许以无偏倚的方式在体内全基因组捕获RNA相互作用。 SPLASH利用体内交联，接近连接和高通量测序来鉴定全球范围内的分子内和分子间RNA碱基配对伙伴。 SPLASH可以应用于不同的生物体，包括细菌，酵母和人类细胞，以及a多样性的细胞条件，以促进理解不同细胞背景下RNA组织的动力学。整个实验SPLASH协议需要大约5天的时间才能完成，计算工作流程需要大约7天的时间才能完成。

图1描述了SPLASH工作流的原理图 。在0.01％的雌二醇和UV交联的情况下加入生物素化的补骨脂素后，从细胞中提取总RNA，并进行斑点印迹，以确保生物素化到RNA的交联有效发生（ 图2 ）。我们使用生物素化的20碱基寡核苷酸作为阳性对照，以滴定待添加到细胞中的生物素化补骨脂素的量，使得每150个碱基中约1个被交联。 随着PCR扩增循环的增加，更多的PCR复制事件会发生，我们使用不同的PCR循环进行小规模的PCR扩增，以确定为深度测序提供足够的材料的最低扩增周期数。在一个有效的图书馆准备过程中，我们是能够在小于15个循环的PCR扩增中从1.5μg大小的所选RNA输入扩增cDNA测序文库（ 图3 ）。然后在高通量测序机上使用2x 150碱基对读数对文库进行测序，然后根据图4中的计算流程对排序读数进行处理。最终结果是包含转录组中分子内和分子间RNA-RNA相互作用的过滤嵌合相互作用的列表（ 表11 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55255/55255fig1.jpg" /> 图1 :SPLASH实验工作流示意图15 。细胞内的成对RNA相互作用在365nm的UV光下使用生物素化的补骨脂素进行交联。交联RNA是then提取并分散到约100个碱基。含有生物素化的补骨脂素交联物的相互作用区域通过与链霉亲和素珠结合并连接在一起而富集。在265nm的UV光下进行反向交联时，将嵌合RNA克隆到cDNA文库中进行深度测序。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55255/55255fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55255/55255fig2.jpg" /> 图2 :通过斑点印迹测试生物素化的交联效率15 。生物素标记的补骨脂素交联RNA 在 1％的雌二醇存在下的体内斑点印迹。不同浓度的交联RNA（20-2,000ng）被点在膜上。不同浓度的生物素化20碱基寡核苷酸被认为是阳性对照。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55255/55255fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55255/55255fig3.jpg" /> 图3: SPLASH的小规模库扩增。将来自RT反应的1μLcDNA用于小规模PCR。反应在10,15和20个循环中取出，并在3％琼脂糖凝胶上运行，以确定文库产生所需的最小扩增循环次数。在该具体实例中，在大规模PCR反应中使用10μLcDNA的10个循环的扩增将产生足够的扩增子进行深度测序。 <img alt="图4" class="xfigimg" src="/files/ftp_upload/55255/55255fig4.jpg" /> <strongclass ="xfig"&gt;图4: SPLASH的计算工作流示意图。从高通量测序的顺序读取被映射到转录组，并使用分析管线对低质量读数，PCR重复，重复映射和剪接连接读取进行过滤。最终输出是表示转录组中分子内和分子间相互作用的嵌合体列表。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55255/55255fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 试剂 </td><td> 体积（μL） </td><td> 最后 </td></tr><tr><td> 无核酸酶自由水 </td><td> 64 </td><td></td></tr><tr><td> 10x T4 PNK缓冲液 </td><td> 8 </td><td> 1X </td></tr><tr><td> 抑制剂20 U /μL） </td><td> 4 </td><td> 1 U /μL </td></tr><tr><td> T4 PNK（10 U /μL） </td><td> 4 </td><td> 0.5 U /μL </td></tr><tr><td> 总 </td><td> 80 </td><td></td></tr></tbody></table> 表1:3&#39;末端修复试剂。 <table fo:keep-together.within-page="1"><tbody><tr><td> 试剂 </td><td> 体积（μL） </td><td> 最后 </td></tr><tr><td> PNK反应 </td><td> 80 </td><td></td></tr><tr><td> 无核酸酶自由水 </td><td> 3 </td><td></td></tr><tr><td> 10x T4 PNK缓冲液 </td><td> 2 </td><td> 1X </td></tr><tr><td> 10 mM ATP </td><td> 10 </td><td> 1mM </td></tr><tr><td> T4 PNK（10 U /μL） </stron克&gt; </td><td>五</td><td> 0.5 U /μL </td></tr><tr><td> 总 </td><td> 100 </td><td></td></tr></tbody></table> 表2:5&#39;末端修复试剂。 <table fo:keep-together.within-page="1"><tbody><tr><td> 试剂 </td><td> 体积（μL） </td><td> 最后 </td></tr><tr><td> PNK反应 </td><td> 100 </td><td></td></tr><tr><td> 10×T4 RNA连接酶缓冲液 </td><td> 6 </td><td> 1X </td></tr><tr><td> 10 mM ATP </td><td> 6 </td><td> 1mM </td></tr><tr><td> R酶抑制剂（20 U /μL） </td><td> 4 </td><td> 0.5 U /μL </td></tr><tr><td> T4 Rnl1（10U /μL） </td><td> 40 </td><td> 2.5 U /μL </td></tr><tr><td> 核酸酶e水</td><td> 4 </td><td></td></tr><tr><td> 总 </td><td> 160 </td><td></td></tr></tbody></table> 表3:邻近结扎试剂。 <table fo:keep-together.within-page="1"><tbody><tr><td> 零件 </td><td> 每次反应量（μL） </td><td> 最后 </td></tr><tr><td> RNA和接头 </td><td>五</td><td></td></tr><tr><td> T4 Rnl2缓冲液（10x） </td><td> 1 </td><td> 1X </td></tr><tr><td> PEG 8000（50％，wt / vol） </td><td> 3 </td><td> 15％（重量/体积） </td></tr><tr><td> R酶抑制剂（20 U /μL） </td><td> 0.5 </td><td> 1 U /μL </td></tr><tr><td> T4 Rnl2（tr）（200U /μL） </td><td> 0.5 </td><td> 10 U /μ，L </td></tr><tr><td> 总 </td><td> 10 </td><td></td></tr></tbody></table> 表4:适配器结扎试剂。 <table fo:keep-together.within-page="1"><tbody><tr><td> 零件 </td><td> 每次反应量（μL） </td><td> 最后 </td></tr><tr><td> 连接和引物 </td><td> 6 </td><td></td></tr><tr><td> 第一链缓冲液（5x） </td><td> 2 </td><td> 1X </td></tr><tr><td> dNTP（10mM） </td><td> 0.5 </td><td> 0.5 mM </td></tr><tr><td> DTT（0.1M） </td><td> 0.5 </td><td> 5 mM </td></tr><tr><td> R酶抑制剂（20 U /μL） </td><td> 0.5 </td><td> 1 U /μL </td></tr><tr><td> 逆转录酶（200U /μL） </s仲&gt; </td><td> 0.5 </td><td> 10 U /μL </td></tr><tr><td> 总 </td><td> 10 </td><td></td></tr></tbody></table> 表5:逆转录试剂。 <table fo:keep-together.within-page="1"><tbody><tr><td> 零件 </td><td> 每次反应量（μL） </td><td> 最后 </td></tr><tr><td> 第一链cDNA </td><td> 6 </td><td></td></tr><tr><td> 单链DNA连接酶缓冲液（10x） </td><td> 1 </td><td> 1X </td></tr><tr><td> 甜菜碱（5 M） </td><td> 2 </td><td> 1 M </td></tr><tr><td> MnCl 2 （50mM） </td><td> 0.5 </td><td> 2.5 mM </td></tr><tr><td> 单链DNA连接酶（100U /μL） </td><td> 0.5 </td><td>五U /μL </td></tr><tr><td> 总 </td><td> 10 </td><td></td></tr></tbody></table> 表6:cDNA的环化试剂。 <table fo:keep-together.within-page="1"><tbody><tr><td> 零件 </td><td> 每次反应量（μL） </td><td> 最后 </td></tr><tr><td> 连接DNA产物 </td><td> 1 </td><td></td></tr><tr><td> 无核酸酶水 </td><td> 10.5 </td><td></td></tr><tr><td> 高保真DNA聚合酶2x主混合物 </td><td> 12.5 </td><td> 1X </td></tr><tr><td> 通用PCR引物（10μM） </td><td> 0.5 </td><td> 0.02μM </td></tr><tr><td> 指数（X）引物（10μM） </td><td> 0.5 </td><td> 0.02μM </tD&gt; </tr><tr><td> 总 </td><td> 25 </td><td></td></tr></tbody></table> 表7:用于小规模PCR的试剂。 <table fo:keep-together.within-page="1"><tbody><tr><td> 步 </td><td> 温度 </td><td> 时间 </td><td> 周期 </td></tr><tr><td> 初始变性 </td><td> 98℃ </td><td> 30秒</td><td> 1 </td></tr><tr><td> 变性 </td><td> 98℃ </td><td> 10秒</td><td></td></tr><tr><td> 退火 </td><td> 65°C </td><td> 30秒</td><td> 25 </td></tr><tr><td> 延期 </td><td> 72°C </td><td> 30秒</td><td></td></tr><tr><td> 最终延期 </td><td> 72°C </td><td> 5分钟</td><tD&gt; 1 </td></tr><tr><td> 保持 </td><td> 4°C </td><td> ∞ </td><td></td></tr></tbody></table> 表8:用于小规模PCR的条件。 <table fo:keep-together.within-page="1"><tbody><tr><td> 零件 </td><td> 每次反应量（μL） </td><td> 最后 </td></tr><tr><td> 连接DNA产物 </td><td>五</td><td></td></tr><tr><td> 无核酸酶水 </td><td> 6.5 </td><td></td></tr><tr><td> 高保真DNA聚合酶2x主混合物 </td><td> 12.5 </td><td> 1X </td></tr><tr><td> 通用PCR引物（10μM） </td><td> 0.5 </td><td> 0.02μM </td></tr><tr><td> 指数（X）引物（10μM） </td><td> 0.5 </td><td> 0.02μM </tD&gt; </tr><tr><td> 总 </td><td> 25 </td><td></td></tr></tbody></table> 表9:用于大规模PCR的试剂。 <table fo:keep-together.within-page="1"><tbody><tr><td> 步 </td><td> 温度 </td><td> 时间 </td><td> 周期 </td></tr><tr><td> 初始变性 </td><td> 98℃ </td><td> 30秒</td><td> 1 </td></tr><tr><td> 变性 </td><td> 98℃ </td><td> 10秒</td><td></td></tr><tr><td> 退火 </td><td> 65°C </td><td> 30秒</td><td> ÿ </td></tr><tr><td> 延期 </td><td> 72°C </td><td> 30秒</td><td></td></tr><tr><td> 最终延期 </td><td> 72°C </td><td> 5分钟</td><td&gt; 1 </td></tr><tr><td> 保持 </td><td> 4°C </td><td> ∞ </td><td></td></tr></tbody></table> 表10:用于大规模PCR的条件。 <img alt="表11" src="/files/ftp_upload/55255/55255tbl11.jpg" /> 表11:计算流水线的分析输出。 "SAM标志拆分读取1"= 0表示读取映射到转录组的正链。 "RNA 1同一性"是指嵌合体左侧的RNA的身份。 "RNA 1起始位置"是指读取沿着RNA 1映射的起始位置。"RNA 1结束位置"是指读取沿着RNA 1映射的最终位置。"Mapping score split reading 1"到映射到RNA 1的读取的映射分数。"Cigar split read 1"指示从读取的"S"剪切的基数和映射到读取"M"的基数。 "Split read 1"表示映射到RNA 1的序列。将同一命名用于嵌合体的右侧，命名为RNA 2. <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55255/55255tbl11large.jpg" target="_blank">请点击此处查看此表的较大版本。</a>

Watch this Scientific Journal Video about Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen at JoVE.com

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

在这里，我们详细介绍了P soralen交联，Ligated和S选择的H ybrids（SPLASH）的S等位基因的方法，使得能够在体内对分子内和分子间RNA-RNA相互作用进行基因组范围的映射。 SPLASH可用于研究包括酵母，细菌和人在内的生物体的RNA相互作用。

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

mapping-rna-rna-interactions-globally-using-biotinylated-psoralen

Nanyang Technological University

Research

JoVE Journal

Genetics

11.9K 次观看.  A*STAR. 在这里，我们详细介绍了P soralen交联，Ligated和S选择的H ybrids（SPLASH）的S等位基因的方法，使得能够在体内对分子内和分子间RNA-RNA相互作用进行基因组范围的映射。 SPLASH可用于研究包括酵母，细菌和人在内的生物体的RNA相互作用。 

视频: Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

科研

遗传学

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

激光局部补骨脂素加合物的单分子分析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs.
The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.

Here, we describe a protocol for genome-wide mapping of the integration sites of Moloney murine leukemia virus-based retroviral vectors in human cells.

逆转录病毒扫描:映射MLV集成站点以定义细胞特异性监管区域

Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

ATAC 基因组在原发性人类 T 淋巴细胞中的染色体全易接近染色质

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

一种快速、定量的后向基因组转化和变异的多肽映射方法

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

Transcription Start Site Mapping Using Super-low Input Carrier-CAGE

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). Accurate detection of TSSs enhances identification and discovery of core promoters. In addition, active enhancers can be detected through signatures of bidirectional transcription initiation. Described here is a protocol for performing super-low input carrier-CAGE (SLIC-CAGE). This SLIC adaptation of the CAGE protocol minimizes RNA losses by artificially increasing the RNA amount through use of an in vitro transcribed RNA carrier mix that is added to the sample of interest, thus enabling library preparation from nanogram-amounts of total RNA (i.e., thousands of cells). The carrier mimics the expected DNA library fragment length distribution, thereby eliminating biases that could be caused by the abundance of a homogenous carrier. In the last stages of the protocol, the carrier is removed through degradation with homing endonucleases and the target library is amplified. The target sample library is protected from degradation, as the homing endonuclease recognition sites are long (between 18 and 27 bp), making the probability of their existence in the eukaryotic genomes very low. The end result is a DNA library ready for next-generation sequencing. All steps in the protocol, up to sequencing, can be completed within 6 days. The carrier preparation requires a full working day; however, it can be prepared in large quantities and kept frozen at -80 &#176;C. Once sequenced, the reads can be processed to obtain genome-wide single-nucleotide resolution TSSs. TSSs can be used for core promoter or enhancer discovery, providing insight into gene regulation. Once aggregated to promoters, the data can also be used for 5&#8217;-centric expression profiling.

Cap Analysis of Gene Expression (CAGE) is a method for genome-wide quantitative mapping of mRNA 5&#8217;ends to capture RNA polymerase II transcription start sites at a single-nucleotide resolution. This work describes a low-input (SLIC-CAGE) protocol for generation of high-quality libraries using nanogram-amounts of total RNA.

使用超低输入载波保持架进行转录启动站点映射

Cap analysis of gene expression (CAGE) is a method used for single-nucleotide resolution detection of RNA polymerase II transcription start sites (TSSs). ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...