Name: enrichment of mrna and bisulfite-mrna library preparation for next-generation sequencing
Uploaded: 2023-07-07T12:00:00.000+00:00
Duration: 6 min 57 s
Description: Watch this Scientific Journal Video about Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study at JoVE.com

这项工作得到了台湾国家科学技术委员会的支持。[NSTC 111-2314-B-006-003]

1. Poly（A） RNA纯化
注:在进行poly（A） RNA纯化之前，使用用DNase I处理的总RNA，并通过毛细管或常规凝胶电泳评估检查总RNA的质量和完整性。研究人员应该能够识别高分子量场中的 28S 和 18S rRNA 核糖体条带以及低分子量场中的 5.8S rRNA 带，而电泳图中没有任何明显的拖尾带。纯化步骤基本上遵循制造商的说明，并在特定步骤中进行了细微的修改。有关本协议中使用的所有材料和仪器的详细信息，请参阅 材料表 。
<ol><li>制备<ol><li>在进行以下程序之前，在室温下预热试剂、洗涤缓冲液、oligo（dT）珠子和裂解缓冲液30分钟。</li>
		<li>为了分离富集poly（A）的RNA，通过将足量的RNA转移到无核酸酶的1.5mL微量离心管中，制备10-20μg高质量总RNA的等分试样作为起始原料。在 70°C 的加热块中孵育5分钟，然后在冰上保持2 分钟。 注:作为起始材料的总RNA的量应根据所需的mRNA量和所用细胞的性质进行适当调整。根据经验，富集poly（A）的RNA可能占总RNA丰度的1-5%。10 μg 高质量总 RNA 的等分试样应提供 100-500 ng 范围内的高质量富集 poly（A） RNA，用于下游亚硫酸氢盐转化。</li>
		<li>同时，完全重悬oligo（dT）磁珠。将所需量的珠子悬浮液转移到新的 1.5 mL 微量离心管中，然后放在磁铁上 3 分钟，直到磁珠形成沉淀。 注:使用 50 μL oligo（dT） 磁珠处理 10 μg 总 RNA 起始量。根据所使用的细胞，可以进一步优化此步骤中的磁珠体积。</li>
		<li>除去上清液，用等量（步骤1.1.3中使用的珠子悬浮液的体积）重悬寡核苷酸（dT）珠子，并在除去上清液之前置于磁铁上3分钟。 注意: 不要过度干燥珠子;用户应快速继续执行步骤 1.2.1。</li>
	</ol></li>
	<li>第一轮净化<ol><li>将裂解缓冲液加入 RNA 样品管（体积比 4:1）并充分混合，然后转移到 oligo（dT） 磁珠中。 注:如果 RNA 样品体积为 20 μL，则加入 80 μL 裂解缓冲液。</li>
		<li>通过移液至少 10 次，将裂解缓冲液混合物和 oligo（dT） 微球完全重悬。</li>
		<li>让样品在室温下连续旋转孵育15分钟。</li>
		<li>将样品放在磁铁上5分钟或直到上清液澄清;弃去上清液。</li>
		<li>向珠子中加入 600 μL 洗涤缓冲液 1，小心混合以重悬珠子。</li>
		<li>将试管放在磁铁上5分钟或直到上清液澄清，弃去上清液，重复洗涤步骤1.2.5一次。</li>
		<li>加入 300 μL 洗涤缓冲液 2 洗涤珠子，并轻轻重悬珠子。</li>
		<li>将试管放在磁铁上5分钟或直到上清液澄清，弃去上清液，重复洗涤步骤1.2.7一次。</li>
		<li>向样品管中加入30μL冷的10mM Tris-HCl（洗脱缓冲液），并在 70°C 下孵育 5分钟。</li>
		<li>快速将试管转移到磁铁上，当悬浮液在20秒或更长时间后澄清时，将含有洗脱的poly（A）富集RNA的上清液转移到新的1.5mL微量离心管中。</li>
	</ol></li>
	<li>第二轮净化<ol><li>向洗脱的RNA样品中加入120μL（洗脱样品体积的4倍）裂解缓冲液并充分混合。</li>
		<li>用等量的裂解缓冲液洗涤第一轮纯化中使用的珠子，如步骤1.1.4，移液10次以重悬珠子，然后将它们放在磁铁上5分钟，然后弃去上清液。</li>
		<li>将RNA:裂解缓冲液混合物转移到洗涤的珠子中，并通过移液至少10x彻底混合。</li>
		<li>将样品在室温下连续旋转孵育15分钟。</li>
		<li>重复步骤1.2.4至1.2.8以除去盐和其他RNA亚型。 注:洗涤缓冲液的体积可减少至 300 μL 洗涤缓冲液 1 和 150 μL 洗涤缓冲液 2。</li>
		<li>向样品管中加入25μL（所需体积）冷的10mM Tris-HCl（洗脱缓冲液），并在 70°C 下孵育 5分钟。</li>
		<li>快速将试管转移到磁铁上，当悬浮液在20秒或更长时间后澄清时，将含有洗脱的poly（A）富集RNA的上清液转移到新的1.5mL微量离心管中。</li>
		<li>将 2.2 μL 转移到 8 联管中，使用 RNA 高灵敏度测定法对 RNA 数量和质量进行质量评估。</li>
		<li>将样品在-20°C下短期储存，在-70°C下储存较长时间。 注意:这可以是协议中的暂停点。</li>
	</ol></li>
</ol>2.亚硫酸氢盐转化
注:离心步骤均在室温下进行。这些程序基本上是按照制造商的说明执行的，但有一个额外的步骤 2.2，用于在亚硫酸氢盐反应步骤 2.3 之前添加加标对照 mRNA 序列。
<ol><li>将 19 μL 纯化的富集 poly（A） RNA 样品转移到 0.2 mL 8 联管中。</li>
	<li>加入1.0μL加标对照，其以预定的1:10,000数量比（即，0.1ng荧光素酶mRNA:1μg纯化的poly（A）富集RNA）无任何修饰）到步骤2.1的RNA样品中。 注:加标对照序列可以是萤火虫荧光素酶RNA、肾荧光素酶或不含甲基化胞嘧啶的 体外 转录RNA序列。</li>
	<li>向试管中加入 130 μL 转化摄政剂，轻轻倒置数次以充分混合。</li>
	<li>将试管放入PCR机中，在70°C下加热10分钟，然后在64°C下孵育45分钟，然后最后一步冷却至4°C。</li>
	<li>将色谱柱放在收集管上，将 250 μL RNA 结合缓冲液转移到色谱柱上。</li>
	<li>将步骤2.4中的样品转移到步骤2.5中的色谱柱中，并彻底移液。</li>
	<li>向色谱柱中加入 400 μL 95-100% 乙醇，迅速盖上盖子，倒置数次。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>向色谱柱中加入 200 μL RNA 洗涤缓冲液。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>加入 200 μL 脱磺缓冲液，在室温下孵育 30 分钟。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>加入400μLRNA洗涤缓冲液，在25°C下以10,000× g 离心30秒，弃去流出物。重复此步骤一次。</li>
	<li>在25°C下再次以10,000× g 离心2分钟，以完全除去残留的洗涤缓冲液。 注:洗脱前的另一个离心步骤2.14 2分钟是完全除去洗涤缓冲液。洗涤步骤执行两次，以洗掉色谱柱上亚硫酸氢盐试剂和脱磺缓冲液中的高盐组分。</li>
	<li>将色谱柱转移到新的 1.5 mL 微量离心管中。</li>
	<li>向色谱柱中加入 20-30 μL 无核酸酶的 H2O，并在室温下静置 1 分钟。</li>
	<li>在25°C下以10,000× g 离心30秒。</li>
	<li>将 2.2 μL 转移到 8 条管中，以评估 RNA 的数量和质量。</li>
	<li>将亚硫酸氢盐处理的RNA样品在-20°C下短期储存，在-70°C下储存较长时间。 注意:这可以是协议中的暂停点。</li>
</ol>3. 亚硫酸氢盐处理的mRNA文库制备
注:按照文库制备说明方案第 4 部分用于纯化的 mRNA 或 rRNA 耗尽的 RNA。第一个启动步骤应遵循 FFPE RNA 方案，因为亚硫酸氢盐处理会使 RNA 片段化。在层流罩中执行每一步，并将反应混合物添加到冰冷冷却架上。
<ol><li>引发起始RNA<ol><li>转移所需量的亚硫酸氢盐处理的 mRNA 样品，总量最大为 5 μL，或加入无核酸酶的 H2O 使总量为 5 μL。 注:建议将至少 10 ng 亚硫酸氢盐处理的 mRNA 作为输入，用于此类 RNA 文库制备并生成 4nM 文库产物的最终摩尔浓度。</li>
		<li>向样品中加入 1 μL 随机引物（丁香），并通过移液混合。</li>
		<li>将样品管置于预设在65°C的PCR机中，并在105°C下盖上盖子5分钟，并在4°C下保持。</li>
	</ol></li>
	<li>第一链cDNA合成<ol><li>向样品中加入 4 μL 合成反应缓冲液（淡紫色）、8 μL 链特异性试剂（棕色）和 2 μL 合成酶混合物（棕色），总体积为 20 μL。 通过移液至少 10 倍并快速旋转彻底混合。</li>
		<li>将样品管置于预设的PCR机中，在25°C下10分钟，42°C下50分钟，70°C下15分钟，并在4°C下保持。 注:对于超过200个碱基的插入物，建议42°C孵育期为50分钟;对于低于200个碱基的插入片段，建议在42°C孵育15分钟。</li>
	</ol></li>
	<li>第二链cDNA合成<ol><li>加入 8 μL 含有 dUTP 混合物（橙色）、4 μL 合成酶混合物（橙色）和 48 μL 无核酸酶 H2O 的合成反应缓冲液，通过移液至少 10 次彻底混合并快速旋转。</li>
		<li>将样品管置于预设在16°C的PCR机中1小时，盖子关闭或≤40°C。</li>
	</ol></li>
	<li>用DNA纯化微球纯化双链cDNA<ol><li>在进行纯化前预热DNA纯化珠30分钟，并涡旋以重悬珠子。</li>
		<li>从步骤3.3.2中向cDNA样品中加入144μL重悬的DNA纯化珠，并通过移液至少10x充分混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后除去上清液并保留含有DNA的珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入带有珠子的试管中，同时将样品保持在磁架上并在室温下孵育 30 秒。</li>
		<li>除去上清液并重复步骤3.4.5一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意: 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架中取出样品，并通过向磁珠中加入 53 μL 0.1x TE（10 mM Tris-HCl 和 1 mM EDTA，pH 8.0）缓冲液来洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁架上5分钟或直到溶液澄清。</li>
		<li>将 50 μL 含有双链 cDNA 的上清液转移到新的 8 条管中。 注意:此时可以停止方案，样品可以储存在-30°C。</li>
	</ol></li>
	<li>cDNA文库的末端制备<ol><li>向洗脱的双链 cDNA 样品中加入 7 μL 末端精纯反应缓冲液（绿色）和 3 μL 末端精纯酶混合物（绿色）。</li>
		<li>以 50 μL 体积设置移液 10 次，在不产生气泡的情况下混合混合物，然后快速旋转样品。</li>
		<li>将样品置于PCR机器中，在20°C孵育30分钟，在65°C孵育30分钟，并在4°C下保持。</li>
	</ol></li>
	<li>接头连接<ol><li>根据制造商的说明稀释适配器（红色）。</li>
		<li>向样品中加入 2.5 μL 稀释的接头、1 μL 连接增强剂（红色）和 30 μL 连接预混液（红色）。</li>
		<li>以 80 μL 体积设置移液 10 次混合混合物，而不会产生气泡，然后快速旋转样品。</li>
		<li>将样品置于PCR机中，并在20°C下孵育15分钟。</li>
		<li>向样品中加入 3 μL 尿嘧啶 DNA 糖基化酶和 DNA 糖基化酶裂解酶核酸内切酶 VIII（蓝色）的混合物并彻底移液。</li>
		<li>将样品放回PCR机中，并在37°C下孵育15分钟，盖子设置为75°C。</li>
	</ol></li>
	<li>纯化连接反应处理的cDNA 注意:使用 SPRIselect 磁珠或可选的 AMpure XP 磁珠的尺寸选择都可用于此目的18.<ol><li>在进行纯化之前预热珠子30分钟，并涡旋以重悬它们。</li>
		<li>将 25 μL 重悬珠转移到步骤 3.6.6 中的 cDNA 样品中，并通过移液至少 10 次彻底混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后将上清液转移到新的8条管中并丢弃珠子。</li>
		<li>向上清液中加入 10 μL 重悬珠，并通过移液至少 10 次彻底混合。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后弃去上清液，不要干扰珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入带有珠子的试管中，同时将样品保持在磁架上并在室温下孵育 30 秒。</li>
		<li>除去上清液并重复步骤3.7.7一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意: 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架中取出样品，并通过向磁珠中加入 17 μL 0.1x TE 缓冲液来洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁架上5分钟，直到溶液澄清。</li>
		<li>将含有靶标末端制备的 dsDNA 的 15 μL 上清液转移到新的 8 条管中。</li>
	</ol></li>
	<li>接头连接DNA的PCR富集<ol><li>向接头连接的 DNA 样品中加入 25 μL 预混液（蓝色）、5 μL 5' 接头引物和 5 μL 3' 接头引物。通过移液至少 10 次彻底混合。</li>
		<li>将样品放入PCR仪中，按照以下步骤进行PCR扩增:（1）98°C30秒，（2）98°C10秒，（3）65°C75秒，（4）65°C5分钟，（5）保持在4°C;其中步骤 （2） 和 （3） 应重复 N+2 循环数，其中 N 等于说明书中的 RNA 输入量。这意味着由于在步骤 3.7 中对样品进行双面尺寸选择，因此需要 2 个额外的循环。 注意:例如，建议在 9-10 个循环的 PCR 中使用 8 ng 纯化的 mRNA 起始量;但在步骤 3.7 中使用双侧、大小选择、连接的 cDNA，推荐的 PCR 循环为 11-12 个循环。</li>
	</ol></li>
	<li>PCR扩增文库的纯化<ol><li>在进行纯化之前，将DNA纯化珠预热30分钟，并涡旋以重悬珠。</li>
		<li>将 45 μL （0.9x） 重悬的珠子转移到步骤 3.8.2 中的 PCR 产物中，并通过移液至少 10x 彻底混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁架上5分钟，直到溶液澄清，然后除去上清液并保留含有DNA的珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入到带有珠子的试管中，将样品留在磁架上，并在室温下静置 30 秒。</li>
		<li>取出上清液并重复步骤3.9.5一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意: 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架上取下样品，向珠子中加入 22 μL 0.1x TE 缓冲液以洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁性架上，静置5分钟或直到溶液在室温下澄清。</li>
		<li>将 20 μL 上清液转移到新的 8 条管中。</li>
		<li>将 2.2 μL 转移到 8 联管中，以评估文库数量和质量。 注:文库的数量也可以通过qPCR检测（参见 材料表）。</li>
		<li>将bsRNA-seq文库样品储存在-20°C。</li>
	</ol></li>
</ol>

创建用于转录组范围作图的稳健亚硫酸氢盐-mRNA 文库

<ol>
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作者没有要披露的利益冲突。

在该协议中，通过使用标准化组件实现了聚（A）富集，亚硫酸氢盐转化和文库制备的详细流程。进一步的测序分析提供了目标样品中mRNA 5-甲基胞嘧啶的鉴定。
关键步骤是起始材料（总RNA）的质量，因为RNA的降解会影响poly（A） RNA纯化的回收率。在进行poly（A） RNA纯化步骤之前，应小心处理样品并避免RNase污染。该过程的另一个关键部分是文库制备中的PCR循环次数。循环数的决定取决于用于文库制备的亚硫酸氢盐处理的mRNA的数量，以及在PCR前的步骤中是使用一种大小的选择还是双大小的选择。因此，目标样品的合适循环数需要试运行，并通过毛细管电泳评估PCR产物大小分布，以确定相同细胞系或组织的最佳循环数。
在该方案中，使用两轮oligo（dT）微珠纯化来富集和纯化poly（A）RNA，并消除大多数核糖体RNA和其他RNA。还有其他方法可以去除核糖体 RNA 并将其他 RNA 类型保留在样品中，例如基于寡核苷酸的 rRNA26 去除。然后，其他RNA类型中存在的5-甲基胞嘧啶修饰也可以用于分析，并扩展对mRNA和其他RNA中5-甲基胞嘧啶特征的理解。
众所周知，亚硫酸氢盐反应无法将 m 5 C 与其他修饰区分开来，包括 5-羟甲基胞嘧啶 （hm5C）、3-甲基胞苷 （m3C） 和 N4-甲基胞苷 （m4C）27。然而，包括 m 5 C RNA甲基转移酶敲低或敲除的实验设计应提供高置信度调节的 m5C 位点的信息证据。此外，使用不同的方法进行验证，例如基于抗体的富集，然后进行测序或PCR或基于LC-MS/MS的方法，基本上可以验证候选的m5C位点16。新兴的直接纳米孔RNA测序技术还通过对当前信号或碱基称为"错误"特征的计算分析提供了RNA修饰的潜在鉴定28,29,30。
Johnson 等人最近的一项研究表明，在三轮亚硫酸氢盐转化后在线粒体 RNA 样品中添加片段化步骤确实显示出更高的转化率并提高了 cDNA 文库产物的产量 31。本文专门分析了线粒体基因组的转化率和读取质量，而不是mRNA转录组。因此， 表1 中更新了片段化步骤的使用，以突出显示已发表的报告是否包括RNA片段化步骤，以便查看者可以轻松复制方案。Zhang等人的另一项研究表明，利用 体外 转录的无修饰RNA文库作为阴性对照可有效消除亚硫酸氢盐处理中的假阳性信号32。最近的研究比较了用于文库构建的不同协议33.用户可以进一步选择在有或没有额外的片段化步骤的情况下进行bsRNA-seq管道，以及 体外 转录的免修饰RNA文库，以定制合适的工作流程。
通过不同的原理对m5C进行全转录组鉴定可以加强修饰图景的发现，并提供对健康或疾病模型中RNA修饰的调控机制的更多理解。

在 150 多种转录后修饰1 中，已在各种类型的 RNA 中鉴定出 5-甲基胞嘧啶 （m5C） 修饰，包括核糖体 RNA、转移 RNA、信使 RNA、micro RNA、长链非编码 RNA、vault RNA、增强子 RNA 和小 cajal 体特异性 RNA2。RNA m 5 C 与多种生物学和病理机制有关，例如调节植物根系发育3、病毒基因表达4 和癌症进展5。该协议的目的是提供简化的管道，以表征处于不同发育阶段或疾病环境中的生物样品的转录组范围的mRNA m5C修饰谱。开发了全转录组亚硫酸氢盐测序来表征亚硫酸氢盐转化 RNA 中碱基对分辨率为 6、7、8、9 的位置和定量胞嘧啶甲基化水平。这在研究 m5C 与参与细胞生物调控机制的基因表达和 RNA 命运的关联时特别有用。在哺乳动物细胞中，有两种已知的 m 5 C 读取器:ALYREF 可以识别细胞核的 m 5 C 并充当 mRNA 细胞核到胞质溶胶转运蛋白10，而 YBX1 可以识别细胞质中的 m5C 并增加 mRNA 稳定性11。系统性红斑狼疮 CD4+ T 细胞中报告了与免疫通路相关的异常 m5C mRNA12。研究表明，mRNA m5C 修饰与癌症免疫调节和癌症进展之间存在关联13,14。因此，在mRNA上绘制m5C修饰图谱可以为阐明潜在的调控机制提供关键信息。
为了研究RNA m 5 C修饰在某些生物学条件下的功能作用，基于亚硫酸氢盐转化（bsRNA-seq）和基于抗体亲和富集的方法，如m 5 C-RIP-seq、miCLIP-seq和5-Aza-seq，可以与高通量测序平台相结合，在转录组范围内使用m5C修饰对靶区和序列进行高效检测15，16.该协议的优势在于，由于基于抗体亲和富集的方法依赖于高质量抗体的可用性，因此该协议的优势在于单碱基分辨率下提供了全面的RNA m 5 C景观，并且可以实现m5C甲基化景观的单片段分辨率17。
所有 RNA 样品都将使用 oligo（dT） 微球进行两轮 mRNA 富集、三个亚硫酸氢盐反应循环和测序文库制备。为了监测 RNA 质量，将在 mRNA 纯化和亚硫酸氢盐反应程序前后通过毛细管凝胶电泳检查每个 RNA 样品，以评估片段分布。纯化的文库将通过其 PCR 扩增子质量进行检查，通过毛细管凝胶电泳检查 DNA 大小分布片段，并在测序前通过基于荧光染料的定量测定检查其总量。该系统还可用于分析广谱生物样品，如农产品、分离的病毒粒子、细胞系、模式生物和病理标本。

<table><tbody><tr><td>Agilent 2100 Electrophoresis Bioanalyzer System</td><td>Agilent, Santa Clara, CA</td><td></td><td>RNA quality detection</td></tr><tr><td>AMpure XP beads</td><td>Beckman Coulter</td><td>A63881</td><td>purify DNA</td></tr><tr><td>Bioanalyzer DNA high sensitivity kit</td><td>Agilent, Santa Clara, CA</td><td>5067-4626</td><td>DNA quality detection</td></tr><tr><td>Bioanalyzer RNA 6000 Pico kit</td><td>Agilent, Santa Clara, CA</td><td>5067-1513</td><td>RNA quality detection</td></tr><tr><td>DiaMag02 - magnetic rack</td><td>Diagenode, Denville, NJ</td><td>B04000001</td><td>assist library preparation</td></tr><tr><td>DiaMag1.5 - magnetic rack</td><td>Diagenode, Denville, NJ</td><td>B04000003</td><td>assist poly(A) RNA purificaion</td></tr><tr><td>Dynabeads mRNA DIRECT purification kit</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>61011</td><td>poly(A) RNA purificaion; Wash Buffer 1 and Wash Buffer 2</td></tr><tr><td>Ethanol</td><td>J.T.Baker</td><td>64-17-5</td><td></td></tr><tr><td>EZ RNA methylation kit</td><td>Zymo, Irvine, CA</td><td>R5002</td><td>bisulfite treatment</td></tr><tr><td>Firefly luciferase mRNA</td><td>Promega, Madison, WI, USA</td><td>L4561</td><td>spike in control seqeunce&amp;nbsp;</td></tr><tr><td>KAPA Library Quantification Kits</td><td>Roche, Switzerland</td><td>KK4824</td><td>library quantification</td></tr><tr><td>Nanodrop spectrophotometer</td><td>Thermo Fisher Scientific, Waltham, MA</td><td></td><td>Total RNA quantity detection</td></tr><tr><td>NEBNext multiplex Oligos for illumina (index Primer set1)</td><td>New England Biolabs, Ipswich, MA</td><td>E7335S</td><td>library preparation</td></tr><tr><td>NEBNext Ultra &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/65352/65352eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; Directional RNA Library Prep Kit for Illumina</td><td>New England Biolabs, Ipswich, MA</td><td>E7760S</td><td>library preparation</td></tr><tr><td>Nuclease-free Water</td><td>Thermo Fisher Scientific</td><td>AM9932</td><td></td></tr><tr><td>P2 pipetman</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>4641010</td><td></td></tr><tr><td>Qubit 2.0 fluorometer&amp;nbsp;</td><td>Thermo Fisher Scientific, Waltham, MA</td><td></td><td>RNA quantity detection</td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>Q32854</td><td>DNA quantity detection</td></tr><tr><td>Qubit RNA HS Assay Kit</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>Q32852</td><td>RNA quantity detection</td></tr></tbody></table>

enrichment of mrna and bisulfite-mrna library preparation for next-generation sequencing

各种类型的 RNA 转录本中的 RNA 转录后修饰与真核细胞中的多种 RNA 调控有关。异常的 RNA 5-甲基胞嘧啶修饰和 RNA 甲基转移酶的失调表达已被证明与包括癌症在内的多种疾病有关。开发了全转录组亚硫酸氢盐测序，以表征亚硫酸氢盐转化的 RNA 在碱基对分辨率下的位置和定量胞嘧啶甲基化水平。在此，该协议详细介绍了两轮poly（A）RNA纯化，三个亚硫酸氢盐反应循环和文库制备的程序，以允许mRNA 5-甲基胞嘧啶修饰位点的转录组范围定位。主反应后RNA数量和质量的评估对于监测RNA完整性至关重要，也是确保高质量测序文库的关键步骤。理想情况下，这些程序可以在三天内完成。通过该方案，使用高质量的总RNA作为输入，实际上可以建立强大的亚硫酸氢盐-mRNA文库，用于从目标样品进行二代测序。

按照本报告中的程序从细胞系19 中生成一系列bsRNA-seq文库 （图1）。在对细胞系样品进行总RNA纯化并进行DNase处理并通过凝胶电泳和紫外-可见分光光度法（A260/A280）检查质量后，RNA样品可以进行poly（A）RNA富集。为了确定双重纯化是否可以去除大部分核糖体RNA，通过毛细管电泳总RNA测定法评估poly（A） RNA的纯化效率，该测定法可以自动计算rRNA污染百分比 （图2）。从RNA片段峰模式和rRNA污染百分比来看，经过两次纯化的RNA样品确实显示，AsPC-1和BxPC-3中核糖体RNA的污染分别从6.5%降低到2.2%和2.6%降低到1.1%。对于人胰腺癌细胞系，两轮微珠纯化可从总 RNA 样品中平均达到 2.8 ± 1% 富含 poly（A） 的 RNA 丰度。因此，通过具有双重纯化步骤的oligo（dT）微球富集poly（A）RNA可最大限度地减少核糖体RNA，并为下游实验提供可行的mRNA样品富集。
亚硫酸氢盐转化方案已在几项关于RNA 5-甲基胞嘧啶修饰的研究中报道（表1）。亚硫酸氢盐反应可以用由40%亚硫酸氢钠和600mM对苯二酚组成的用户复溶反应混合物在水溶液（pH 5.1）中于75°C下进行4小时10,20,21。或者，也可以使用商业试剂盒对 RNA 样品进行更严格的亚硫酸氢盐处理;在其他人和我们的许多研究中，6,22,23,24 将亚硫酸氢盐反应时间延长到三个孵育周期。由于三周期孵育步骤在体外转录和结构上折叠更多的大肠杆菌 16S rRNA 片段8 中有效地转化未甲基化的胞嘧啶，因此在该方案中也应用了三周期孵育步骤。
通过两次poly（A）富集和三循环亚硫酸氢盐转化，通过毛细管电泳评估反应前后的RNA质量。由于亚硫酸氢盐反应引起的碎裂，亚硫酸氢盐处理后的RNA大小分布显示出~200-500个核苷酸的峰值（图3）。此外，片段化的 bsRNA 是文库制备的理想选择，无需进行另一个片段化步骤。总共使用8-10ng的bsRNA作为输入，通过在接头连接DNA的最终PCR扩增中使用9至11个循环来根据该方案构建文库。扩增子的毛细管电泳显示文库制备相当成功，仅剩下一小部分引物，没有过扩增峰（图4）。为了检查每个样品库中的转化效率，在进行亚硫酸氢盐处理之前，将未甲基化的萤火虫荧光素酶RNA作为加标对照序列加入样品中。使用meRanTK工具包25将测序读数与参考序列比对后，将总读长的平均值2,440（0.006%）映射到加标序列，分析的总C-to-T转化率达到平均99.81%;可以在集成基因组学查看器中查看状态（图5）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65352/65352fig01.jpg"> 图1:亚硫酸氢盐mRNA测序的工作流程图。该流程由三个主要方案组成，包括 mRNA 富集、亚硫酸氢盐转化和文库制备。缩写:bsRNA = 亚硫酸氢盐 mRNA;质量控制。= 质量控制。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65352/65352fig02.jpg"> 图2:毛细管电泳富集poly（A）的RNA样品的质量控制结果。（A） BxPC-3 细胞总 RNA 的代表性毛细管电泳图谱。 （B ） AsPC-1 和 BxPC-3 细胞中经寡核苷酸 （dT） 纯化处理的样品的代表性毛细管电泳曲线。mRNA E1，一次性纯化的mRNA洗脱;mRNA E2，mRNA洗脱来自两次纯化。rRNA污染估计值由控制总RNA分析测定的电泳操作系统计算。 （C） 分子量标准物的毛细管电泳曲线是在同一次运行中获得的，样品如图 B 所示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65352/65352fig03.jpg"> 图3:通过毛细管电泳对RNA样品进行质量评估。（A）总RNA，（B）来自两次纯化的poly（A）富集mRNA，以及（C）来自BxPC-3细胞的亚硫酸氢盐处理的mRNA的代表性毛细管电泳曲线。 缩写:B_empty = 用空载体转导的 BxPC-3 细胞;B_shScr = 用加扰序列转导的 BxPC-3 细胞。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65352/65352fig04.jpg"> 图4:bsRNA-seq文库的质量评估。（空调） 使用来自一组 BxPC-3 细胞的 bsRNA，通过 PCR 以 9 和 11 个循环的不同 PCR 周期数扩增构建的 bsRNA-seq 文库的三组代表性毛细管电泳图谱。通过高灵敏度RNA定量测定法确定亚硫酸氢盐处理的RNA的估计起始量。 （A ） BxPC-3模拟样品、（ B ） BxPC-3空样品和 （C） BxPC-3 shN2UN2样品中分别使用了8、8.4和9.6 ng bsRNA。分子量标准标记物峰为35 bp，10,380 bp代表每个样品电泳曲线中下边界和上限的内标。分别有约100 bp的突出峰和约127 bp的模糊峰，表明PCR引物（<100 bp）和接头二聚体（~127 bp）在PCR纯化后仍留在洗脱液中。缩写:BxPC-3 模拟 = 未转导的 BxPC-3 细胞;BxPC-3 空 = 用空载体对照转导的 BxPC-3 细胞;BxPC-3 shN2UN2 = 用 shNSUN2 构建体转导的 BxPC-3 细胞。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65352/65352fig05.jpg"> 图 5:映射到对照刺突参考序列的每个 bsRNA-seq 文库的序列比对曲线。 BxPC-3 模拟文库、空文库、加扰文库和 shNSUN2 bsRNA-seq 文库与萤火虫荧光素酶刺突对照参考序列（表示为"Z"基因）的代表性比对谱;显示了 2 个具有代表性的区域。灰色条表示在参考序列的指示碱基位置呈现匹配的共有核苷酸的所有读段。红色条突出显示读数底部位置的胸苷 （T） 身份;蓝色条表示在该位置具有胞嘧啶 （C） 同一性的读数。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352fig05large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
表1:亚硫酸氢盐反应方案和转化率的比较。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65352/65352_JoVE_Table1%20(1).xlsx" target="_blank">请按此下载此表格。</a>

Watch this Scientific Journal Video about Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study at JoVE.com

作者聚焦:解码 RNA 甲基化在胰腺癌中的作用 - 一项单碱基分辨率研究 

该协议提供了一种易于遵循的工作流程，可使用标准化设备对感兴趣的生物样品进行poly（A）RNA纯化、亚硫酸氢盐转化和文库制备。

mRNA和亚硫酸氢盐-mRNA文库的富集制备用于二代测序

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA ...

本研究旨在研究 RNA 甲基化，特别是 5-甲基胞嘧啶修饰在胰腺癌中的作用。我们想要回答的问题是某些RNA甲基转移酶的上调或下调如何影响癌细胞表型，以及RNA底物通过哪些表型。本研究使用标准化设备以单碱基分辨率提供全面的 RNA m5C 图景。从这项使用亚硫酸氢盐RNA seq的研究中，结果将RNA m5C修饰与癌细胞分化不良的关联联系起来。这应该为未来的研究铺平道路，以辨别RNA转录本中特定m5C的乘客与疾病驱动因素的影响。通过这种技术，我们可以识别可能参与肿瘤进展的一组差异甲基化RNA。此外，单分子碱基实验可以提供特异性RNA m5C的更详细的调控作用。

enrichment-mrna-bisulfite-mrna-library-preparation-for-next

Nanyang Technological University

Research

JoVE Journal

Biology

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

1.0K 次观看.  National Cheng Kung University. 本研究旨在研究 RNA 甲基化，特别是 5-甲基胞嘧啶修饰在胰腺癌中的作用。我们想要回答的问题是某些RNA甲基转移酶的上调或下调如何影响癌细胞表型，以及RNA底物通过哪些表型。本研究使用标准化设备以单碱基分辨率提供全面的 RNA m5C 图景。从这项使用亚硫酸氢盐RNA seq的研究中，结果将RNA m5C修饰与癌细胞分化不良的关联联系起来。这应该为未来的研究铺平道路，以辨...

视频: 作者聚焦:解码 RNA 甲基化在胰腺癌中的作用 - 一项单碱基分辨率研究 

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

科研

遗传学

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

<meta charset="utf8"></head><body>2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS Seq:一种基于 LC-MS 的通用方法，用于对具有不同核苷酸修饰的 RNA 混合物进行直接和从头测序

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

使用 AQRNA-seq 分析 tRNA 动力学

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

作者聚焦:AQRNA-seq 在绘制小 RNA 和解开蛋白质翻译机制中的作用

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

用于定量小 RNA 的 AQRNA-seq

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

细菌中的RIBO-seq:NGS测序样本收集和库准备协议

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

生物化学

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

发育生物学

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

单次读取和配对的最终从10微克总RNA的mRNA - Seq的Illumina的图书馆

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

mRNA和亚硫酸氢盐-mRNA文库的富集制备用于二代测序

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用于定量小 RNA 的 AQRNA-seq

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用于定量小 RNA 的 AQRNA-seq

细菌中的RIBO-seq:NGS测序样本收集和库准备协议

为早期小鼠胚胎测序准备小型RNA库

单次读取和配对的最终从10微克总RNA的mRNA - Seq的Illumina的图书馆