We thank all the authors of the original ERRBS report. We thank Mame Fall for technical assistance. We acknowledge the Weill Cornell Medical College Epigenomics Core for technical services and assistance. The work was supported by a Sass Foundation Judah Folkman Fellowship, an NCI K08CA169055 and ASH-AMFDP12005 to FGB, NIH R01HG006798 and R01NS076465, funding from the Irma T. Hirschl and Monique Weill-Caulier Charitable Trusts and STARR Consortium (I7-A765) to CEM, and an LLS SCORE grant (7006-13) to AMM.

执行所有的程序都是由印第安纳大学医学院实验动物管理和使用委员会的批准，并按照卫生指引研究院。 1.手术技巧<ol><li>这个过程通过使用无菌手套，仪器，并根据美国国立卫生研究院指南25的无菌外科领域中保持无菌技术。消毒高压灭菌他们开始在手术前的工具（ 见表特定试剂/设备的完整列表）。使用玻璃珠灭菌的操作过程中，以消毒的工具。 </li></ol> 2.麻醉和准备<ol><li>麻醉小鼠在麻醉箱以0.9升/分钟的氧气和2.5％的异氟醚用兽医异氟烷蒸发器系统的混合物。确保鼠标并没有改变身体姿势从包装盒中取出前作出回应。 </li><li>应用眼药膏谅解备忘录SE的眼睛以保护他们免受干燥。 </li><li>从箱气流切换到鼻锥。将鼠标正视在其左侧上的加热垫覆盖有手术垫和吸收长凳纸与它的鼻子和嘴的锥体内。连续监测小鼠的呼吸节奏和速​​度，并调整异氟烷水平根据需要（2.5之间 - 3％异氟醚）以维持麻醉的适当水平，并且使用脚趾捏反射来确认总镇静。 </li></ol> 3.手术方法<ol><li>对齐并与外科领域关注的实体镜。调整鼻锥和磁带下来，以便它位于沿视野的边缘。 </li><li>用鼠标卧在其左侧，用胶带将右耳到鼻锥体的边缘，在那里露出的切口将作出在耳后的区域。确保耳后静脉水平会穿过耳朵。需要注意的是T的正确位置他的动物和耳朵的录像都是为了快速找到面神经的关键。 </li><li>润湿毛发上，并用70％乙醇在耳朵后面，刮胡子使用剃刀或手术刀刀片的外科手术部位。预湿的皮草，使剃须在这个解剖位置比较容易。 </li><li>清洁与碘溶液，如聚维酮碘的外科擦洗（7.5％聚维酮碘），随后用70％乙醇的皮肤。重复该清洗2次以上，以彻底消毒的区域。 </li><li>确定在何处作出切口，从耳尾端跟踪耳后静脉的区域后方的耳突起。使用弹簧剪刀，制作4毫米的切口2 - 3毫米后的突起。 </li><li>通过解剖用钝性分离皮下脂肪和筋膜。避免直接切削用剪刀因为血管或肌肉组织很容易损坏。 </li><li>如果发生出血，将压力施加到手术部位用无菌棉签为至少30秒。如果发生显著流体损失，以达到的用25或27G的针的无菌0.9％盐溶液0.5毫升腹膜内注入小鼠。 </li><li>使用几个键标，副神经，耳道，和前二腹肌（下面描述），以定位面神经。剖析围绕这些地标，直到面神经的分支可视化。当它被发现的神经将显示为显著固体白色组织和一层筋膜附着到下面的结构。 <ol><li>发现副神经，从头骨的尾部部分，其行进到支配斜方肌，一旦皮下脂肪和筋膜已解剖。面神经是深副神经。 </li><li>发现软骨耳道看起来珍珠白，并且可以看出延髓面神经。 </li><li>发现前二腹肌的肌腹的位于顶部和caudal面神经。 </li></ol></li><li>如果面神经的主要分支的可视化，跟踪他们背来找到自己的出身，从茎乳孔。用细尖杜蒙钳＃四十五分之五保持手术部位的开放，推进下面的神经路径的春天剪刀的提示，然​​后将镊子背侧，以保持新的先进地区开放。 </li><li>可视面神经与颧骨，颊的躯干，并且在这一点上下颌缘分支。 注:颞支会发现接近孔。边际下颌神经分支到其上，下部分更靠近钳口，从而那些神经分支将不会在这个水平可见的。 <ol><li>如果执行神经切断，轻轻稳定神经的精尖钳和切割​​神经与弹簧剪刀。避免将过多的牵引神经与血管钳，以防止avulsing从脑干神经。推树桩相互远离，或切割并移除远端神经的一部分，以确保没有重新连接可以发生。 </li><li>如果执行一个挤压伤，利用蒙特＃四十五分之五镊子压缩神经为30秒，使用恒定压力以切断所有轴突，则在垂直于第一粉碎部位的第二角度重复此压碎。避免将压力可变数量的30秒美眉期间，否则伤害将是动物之间的不一致。 </li></ol></li></ol> 4.关闭和恢复<ol><li>重新定位的脂肪和肌肉在底层结构。 </li><li>近似​​切口的边缘，并使用7.5毫米伤口夹闭合伤口。缝线或胶水还可以接受的伤口闭合。手术后止痛药可以在此时提供。 </li><li>取下鼠标的耳朵磁带。关掉异氟烷流动，并允许小鼠呼吸纯氧为30秒至1分钟。 PL王牌鼠标在一个空笼没有被褥从麻醉中恢复。 </li><li>当鼠标被回收，检查其面瘫的验证标志的行为。胡须就会瘫痪和角度背对着脸颊，鼻子就会偏离，并且眼睛也不会因空气的粉扑闪烁。 </li><li>房子动物联合手术后，如果他们是女性。避免住房雄性小鼠联合，因为他们更积极，而且往往强行删除其cagemate的伤口剪辑，从而导致感染。如有必要，提供手术后止痛剂，此时，。 </li><li>监测小鼠，每天一次，连续几天的操作后，以确保没有感染或其他并发症发生术后。手术后10天，如果他们没有倒下了自己 - 删除伤口片段7。 </li><li>适用于润滑眼膏，以患眼每天以防止角膜并发症，无论是直到眨眼反射是重覆盖或直到安乐死。 </li></ol>

作者有没有利益冲突披露。

胞嘧啶甲基化在生物相关的基因组区域的协议收益率呈现碱基对分辨率的数据。作为写入的50纳克原料优化的协议，但是，它可以适用于处理各种输入材料（5纳克或多个）26。这将需要的一些协议的步骤的调整，如表6所示。该ERRBS库适合于配对末端测序和进一步的基因组的覆盖，也可通过测序完成读长于51个周期。多路复用测序将提供每个样品以较低的成本协议，然而，这将导致每个中的数据（ 图5和表8）表示CpG部位减少的覆盖范围，并且不会产生覆盖足够的深度来执行需要每高覆盖率的分析CpG部位（ 如所描述的Landan 等人33）。最后，这个协议（或任何亚硫酸氢基protocoL）不能甲基胞嘧啶和羟甲基胞嘧啶46,47之间的区别。然而，生成可与其它协议被集成的数据结果48,49划定的不同的修改，以及其他的胞嘧啶修饰最近报道50，它们应的兴趣。 高品质的库将出现如图3A-C，以及一旦合并用于测序产生了跟踪， 如图3G（红色迹线），代表来自两个文库的馏分等摩尔贡献。文库制备故障可导致在操作过程中的任何步骤。如果降解的DNA进行处理，将导致在使用在此协议中描述的测序参数不富含MspI酶切位点的片段，从而在低的CpG覆盖库。如果一种酶是无功能的或无意排除在反应之一，该协议将不会产生预期的库。如果结扎意图ction是低效的，适配器是在更高的浓度比预期的，和/或使用的引物浓度是限制性试剂的最终扩增步骤，库可能会发生故障。过量适配器（视为在生物分析结果的峰在〜150bp的图 ; 图3D-F），在库中还会干扰测序由于既库和过量的适配器的滥聚类。而这样的库可能顺序显然通常，的一个显著部读出将是仅仅接头序列。如果过量适配器库中观察到的，这是最好的，以重复文库制备，如果使用优化的输入材料到适配器数量比率的材料是可用的。最后，为了确保库的有效的PCR扩增，所述较低和较高文库级分被保持在整个重亚硫酸盐转化和PCR富集步骤分离的样品。如果不这样做会产生扩增的P期间差分效率较高和较低的级分的CR反应（如在图3G蓝色迹线所示）和用于各个基因组位点的测序过程中涉及的每个库馏分不等代表性的潜力。用户可以选择包括亚硫酸氢盐转化为以扩增而产生的库需要最佳PCR循环进一步滴定后定量PCR立即步骤。 ERRBS文库制备协议有其特定的试剂推荐几个关键步骤。在最终修复工序中，使用了四核苷酸dNTP混合物的允许端修复的不含CG突出端，如那些从MspI酶切位点的酶星活性和存在于原始DNA样品中剪切的DNA片段得到的任何产品。这导致在结果改进的CpG表示。在连接步骤，以确保关键是要使用高浓度的连接酶（2000000单位/ ml）和甲基适配器结扎reacti上是有效的，该亚硫酸氢盐转化不影响对于准确的数据对准所必需的接头序列。在PCR步骤中，使用能够扩增的亚硫酸氢处理的富含GC的DNA片段的聚合酶是必要的高特异性。最后，为了保证消除多余适配器和引物，SPRI珠粒纯化（例如:Agencourt AMPure XP）被推荐，而不是基于列的测定法用于连接和PCR产物隔离。 为了生成高质量的数据时，它以确保有效的亚硫酸氢盐转化是重要的。提出的控制提供了用户确定测序前的转换效率的能力。作为替代，非人类的DNA，例如λ噬菌体DNA可以用作内部对照（尖峰式）。由于品种的差异，这种类型的控件，可以直接计入下游测序（ 如所使用的玉等人。34）。然而，如果尖峰在我š利用，它不能被用来确定前文库测序转换效率，除非唯一地放大，并独立地为之前文库测序测序。确定的转换率是基于在非CpG位点的甲基化状态。这可能不适合于在高的胞嘧啶甲基化非CpG上下文（例如胚胎干细胞）和平行的样品或评估可以用于此目的的转换效率的其它手段的情况下使用。 有几个注意事项，以地址是唯一的ERRBS库的顺序。测序的文库级分的前三个碱基为几乎均匀地非随机由于MspI酶切位点的识别剪切位点（C ^ CGG;参见图4B，C）。这导致了显著数据丢失的可能性，由于低质量读取从排序过程中，尽管明显高集群密度差集群的定位所致。为了克服这个障碍，包括一个独立的车道（PhiX管制或其他库型）作为专用车道控制在一个高度复杂的图书馆。高度复杂的图书馆有包含在测序的前四个基地的A，C，T和G均衡的代表性结束。合适的对照泳道包括例如RNA-SEQ，芯片起，全基因组测序，或通过测序机床制造商（ 例如 PhiX控制V3）提供一个控制库。当被指定为一个对照泳道用于各个测序运行，它可以作为基础，它在测序前四个碱基用来检测簇的位置的矩阵生成。高品质的读取捕获将提高每个CpG位点的平均覆盖5.2（N = 4）。可替代地，本技术难度也可以使用暗测序方法如先前所述23克服。其他测序标准遵循每制造商的协议标准操作程序。最后，每个CpG的C中的覆盖面Hosen的用于数据分析将被用户和部分被引导通过感兴趣的生物的问题。 10倍覆盖范围的阈值提供了一个高覆盖率的分析方法，但是这个门槛可以降低应该是这样的兴趣。 ERRBS数据分析的完整讨论超出了本文的范围，但是，差异甲基化的胞嘧啶和地区可以使用开源工具31,51-53确定。额外的分析考虑和方法已被充分描述54,55，读者被鼓励以搜索文献为最适合于计划 ​​的分析工具。 相比其他公布方法，ERRBS提供，当为可重复性的描述产量高利率进行了为期四天的协议。相比黄金标准MassARRAY EpiTYPER 26已经验证，具有成本效益的高覆盖率的数据，并适用于各种输入材料量（有利低频的临床样品的处理和其他类型的细胞）和测序的方法。它提供了碱基对分辨率在生物学相关基因座，可以在综合可用于其它技术分析基因组范围的转录因子结合，染色质重塑，表观遗传标记和其他感兴趣的胞嘧啶修饰的分析。在这些研究中ERRBS数据利用可以向一个全面的分子的方法，并允许在生物模型和人类疾病的研究高维分析。

在胞嘧啶（5-甲基胞嘧啶）DNA甲基化是一个后生标记为各种生物过程中哺乳动物细胞至关重要的，包括但不限于印记，X染色体失活，发展和基因表达1-8的调节。的DNA甲基化模式在恶性和其它病症的研究已经确定疾病特定图案，并有助于疾病的发病机制和可能的生物标志物的发现9-17的理解。有迹象表明，询问表观基因组DNA甲基化状态的许多协议。这些可以分为基于亲和力的，限制性内切酶为基础的，而利用微阵列或测序平台下游重亚硫酸盐转化为基础的分析。此外，也有弥补这些一般类别，包括但不限于，结合亚硫酸氢盐限制性分析18和精简表示亚硫酸氢盐测序（RRBS 19）几个协议。 </P&gt; RRBS最初由迈斯纳等人 19,20说明。该协议引入了一个步骤，以丰富随后亚硫酸氢盐测序，结果定量碱基对分辨率的数据是符合成本效益21,22富含GC的基因组区域。所述富含GC的区域由MspI酶切位点（C ^ CGG）限制性内切酶有针对性的，和胞嘧啶甲基化是由胞嘧啶的亚硫酸氢盐转化率（未修饰的胞嘧啶到尿嘧啶脱氨），随后通过聚合酶链式反应（PCR）扩增得到解决。 RRBS覆盖大多数基因的启动子和CpG岛中所需的全基因组测序的一小部分;然而RRBS的CpG了海岸和生物相关的其他间隔区的覆盖范围有限。因为这改善后，这些基因组区域23-25 ​​的方法和产生的覆盖原始报表几组已发布更新RRBS协议。增强的减少代表色曲亚硫酸氢接近开关就是嵌入式（ERRBS）包括相比RRBS库准备修改和替代数据对齐方式26。 ERRBS导致数生成的数据所代表的CpG更高，增加了覆盖范围的所有基因组区域的审问26。这种方法已被用来解决在人类患者和其它动物标本26-30 DNA甲基化模式。 所述ERRBS协议上描述所需完成和使用代表人类的DNA所产生的数据的所有步骤提供的信息（样品从先前报道，去识别患者样品31得到的，并从一个正常人供体的CD34 +骨髓样品）。该协议包括一个自动大小选择过程，从而降低了处理时间，每个样品，并允许在文库大小选择提高精度。该协议结合了一连串的既定的分子生物学技术。高分子量DNA被消化瓦特第i个甲基化敏感限制性内切酶（MspI酶切位点），其次是年底修复，A-拖尾，以及甲基化的适配器连接。所述富含GC的片段的大小选择之后是亚硫酸氢盐转化率和之前测序PCR扩增。亚硫酸氢盐转化一直是前面所述32和数据分析和应用程序的详细审查超出了本文的范围，但是建议和参考文献包含了读者使用。该协议可以四天执行，并且是适合于小输入（50毫微克或更少）的材料的量。该协议为每个CpG位点不仅对于差分甲基化位点和区域确定而且对后生多态性检测足够高覆盖率描述产量数据所描述的Landan 等人 33。

<table border="0" cellpadding="0" cellspacing="0" width="1216">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Name of Reagent/ Equipment</td>
			<td style="width:153px;">Company</td>
			<td style="width:104px;">Catalog Number</td>
			<td style="width:193px;">Comments/Description</td>
			<td style="width:497px;">URL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">MspI</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">R0106M</td>
			<td style="width:193px;">100,000 units/ml</td>
			<td style="width:497px;">https://www.neb.com/products/r0106-mspi</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">NEBuffer 2</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">B7002S</td>
			<td style="width:193px;">Reaction buffer for MspI enzyme; protocol step 1.2</td>
			<td style="width:497px;">https://www.neb.com/products/b7002-nebuffer-2</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:269px;">Phenol solution</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:104px;">P4557</td>
			<td style="width:193px;">Equilibrated with 10&#160;mM Tris HCl, pH&#160;8.0; see safety and handling instructions at http://www.sigmaaldrich.com/catalog/product/sigma/p4557</td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sigma/p4557</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:269px;">Chloroform</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:104px;">C2432</td>
			<td style="width:193px;">See safety and handling instructions at http://www.sigmaaldrich.com/catalog/product/sial/c2432</td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sial/c2432</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Glycogen</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:104px;">G1767</td>
			<td style="width:193px;">19-22 mg/ml</td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sigma/g1767</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">NaOAc</td>
			<td>Sigma-Aldrich</td>
			<td>S7899</td>
			<td style="width:193px;">3M pH 5.2</td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sigma/s7899</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Ethanol</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:104px;">E7023</td>
			<td style="width:193px;">200 proof, for molecular biology</td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sial/e7023</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Buffer EB</td>
			<td style="width:153px;">Qiagen</td>
			<td style="width:104px;">19086</td>
			<td style="width:193px;">10 mM Tris-Cl, pH 8.5</td>
			<td style="width:497px;">http://www.qiagen.com/products/catalog/lab-essentials-and-accessories/buffer-eb</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">tris(hydroxymethyl)aminomethane (Tris)</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:104px;">T1503</td>
			<td style="width:193px;">prepare a 1M pH 8.5 solution</td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sigma/t1503</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Tris- Ethylenediaminetetraacetic acid (TE)</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:104px;">T9285</td>
			<td style="width:193px;">Dilute to 1X buffer solution per manufacturer&#39;s recommendations</td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sigma/t9285</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">T4 DNA Ligase Reaction Buffer</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">B0202S</td>
			<td style="width:193px;">10X concentration</td>
			<td style="width:497px;">https://www.neb.com/products/b0202-t4-dna-ligase-reaction-buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Deoxynucleotide triphosphate (dNTP) Solution Mix</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">N0447L</td>
			<td style="width:193px;">10 mM each nucleotide</td>
			<td style="width:497px;">https://www.neb.com/products/n0447-deoxynucleotide-dntp-solution-mix</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">T4 DNA Polymerase</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">M0203L</td>
			<td style="width:193px;">3,000 units/ml</td>
			<td style="width:497px;">https://www.neb.com/products/m0203-t4-dna-polymerase</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">DNA Polymerase I, Large (Klenow) Fragment</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">M0210L</td>
			<td style="width:193px;">5,000 units/ml</td>
			<td style="width:497px;">https://www.neb.com/products/m0210-dna-polymerase-i-large-klenow-fragment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">T4 Polynucleotide Kinase</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">M0201L</td>
			<td style="width:193px;">10,000 units/ml</td>
			<td style="width:497px;">https://www.neb.com/products/m0201-t4-polynucleotide-kinase</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:269px;">QIAquick PCR Purification Kit</td>
			<td style="width:153px;">Qiagen</td>
			<td style="width:104px;">28104</td>
			<td style="width:193px;">Used for DNA product purification in protocol step 2.3</td>
			<td style="width:497px;">http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/dna-cleanup/qiaquick-pcr-purification-kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">2&#39;-deoxyadenosine 5&#39;-triphosphate (dATP)</td>
			<td style="width:153px;">Promega</td>
			<td style="width:104px;">U1201</td>
			<td style="width:193px;">100 mM</td>
			<td style="width:497px;">http://www.promega.com/products/pcr/routine-pcr/deoxynucleotide-triphosphates-_dntps_/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Klenow Fragment (3&#39;&#8594;5&#39; exo-)</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">M0212L</td>
			<td style="width:193px;">5,000 units/ml</td>
			<td style="width:497px;">https://www.neb.com/products/m0212-klenow-fragment-3-5-exo</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:269px;">MinElute PCR Purification Kit</td>
			<td style="width:153px;">Qiagen</td>
			<td style="width:104px;">28004</td>
			<td style="width:193px;">Used for DNA product purification in protocol step 3.3</td>
			<td style="width:497px;">http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/dna-cleanup/minelute-pcr-purification-kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">T4 DNA Ligase</td>
			<td style="width:153px;">New England Biolabs</td>
			<td style="width:104px;">M0202M</td>
			<td style="width:193px;">2,000,000 units/ml</td>
			<td style="width:497px;">https://www.neb.com/products/m0202-t4-dna-ligase</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Methylation Adapter Oligo Kit</td>
			<td style="width:153px;">Illumina</td>
			<td style="width:104px;">ME-100-0010</td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:269px;">Agencourt AMPure XP</td>
			<td style="width:153px;">Beckman Coulter</td>
			<td style="width:104px;">A63881</td>
			<td style="width:193px;">Used in protocol sections that implement magnetic bead purification steps (steps 4.3 and 8.2). Equilibrate to room temperature before use</td>
			<td style="width:497px;">https://www.beckmancoulter.com/wsrportal/wsrportal/wsr/research-and-discovery/products-and-services/nucleic-acid-sample-preparation/agencourt-ampure-xp-pcr-purification/index.htm?i=A63882#2/10//0/25/1/0/asc/2/A63882///0/1//0/</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">Pippin Prep Gel Cassettes, 2% Agarose, dye-free</td>
			<td style="width:153px;">Sage Science</td>
			<td style="width:104px;">CDF2010</td>
			<td style="width:193px;">with internal standards</td>
			<td style="width:497px;">http://store.sagescience.com/s.nl/it.A/id.1036/.f</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Certified Low Range Ultra Agarose</td>
			<td>Bio-Rad</td>
			<td>161-3106</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.bio-rad.com/en-us/sku/161-3106-certified-low-range-ultra-agarose</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-Borate-EDTA (TBE) buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T4415</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sigma/t4415</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethidium bromide solution</td>
			<td>Sigma-Aldrich</td>
			<td>E1510</td>
			<td style="width:193px;">10 mg/ml</td>
			<td style="width:497px;">http://www.sigmaaldrich.com/catalog/product/sigma/e1510</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 bp DNA Ladder</td>
			<td>NEB</td>
			<td>N3236S</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">https://www.neb.com/products/n3236-50-bp-dna-ladder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 bp DNA Ladder</td>
			<td>NEB</td>
			<td>N3231S</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">https://www.neb.com/products/n3231-100-bp-dna-ladder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gel Loading Dye, Orange (6X)</td>
			<td>NEB</td>
			<td>B7022S</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">https://www.neb.com/products/b7022-gel-loading-dye-orange-6x</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scalpel&#160;Blade&#160;No.&#160;11</td>
			<td>Fisher Scientific</td>
			<td>3120030</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.fishersci.com/ecomm/servlet/fsproductdetail?position=content&#38;tab=Items&#38;productId=11876776</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.qiagen.com/products/catalog/sample-technologies/dna-sample-technologies/dna-cleanup/qiaquick-gel-extraction-kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">EZ DNA Methylation Kit</td>
			<td style="width:153px;">Zymo Research</td>
			<td style="width:104px;">D5001</td>
			<td style="width:193px;">Used in protocol step 6.2</td>
			<td style="width:497px;">http://www.zymoresearch.com/epigenetics/dna-methylation/bisulfite-conversion/ez-dna-methylation-kits/ez-dna-methylation-kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">EZ DNA Methylation-Lightning Kit</td>
			<td style="width:153px;">Zymo Research</td>
			<td style="width:104px;">D5030</td>
			<td style="width:193px;">Alternative for step 6.2</td>
			<td style="width:497px;">http://www.zymoresearch.com/epigenetics/dna-methylation/bisulfite-conversion/ez-dna-methylation-lightning-kit</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:269px;">Universal Methylated Human DNA Standard</td>
			<td style="width:153px;">Zymo Research</td>
			<td>D5011</td>
			<td style="width:193px;">Used as bisulfite conversion control</td>
			<td style="width:497px;">http://www.zymoresearch.com/epigenetics/dna-methylation/methylated-dna-standards/universal-methylated-human-dna-standard</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">FastStart High Fidelity PCR System</td>
			<td style="width:153px;">Roche</td>
			<td style="width:104px;">03553426001</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://lifescience.roche.com/shop/products/faststart-high-fidelity-pcr-system#tab-0</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:269px;">Qubit dsDNA High Sensitivity Assay Kit</td>
			<td style="width:153px;">Life Technologies</td>
			<td style="width:104px;">Q32854</td>
			<td style="width:193px;">A fluorescence-based DNA quantitation assay; used in protocol steps 1.1, 9.1 and 10.1</td>
			<td style="width:497px;">http://www.lifetechnologies.com/order/catalog/product/Q32854</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">DynaMag-2 Magnet</td>
			<td style="width:153px;">Life Technologies</td>
			<td>12321D</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">https://www.lifetechnologies.com/order/catalog/product/12321D</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">High Sensitivity DNA Kit</td>
			<td style="width:153px;">Agilent Technologies</td>
			<td style="width:104px;">5067-4626</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.genomics.agilent.com/en/Bioanalyzer-DNA-RNA-Kits/High-Sensitivity-DNA-Analysis-Kits/?cid=AG-PT-105&#38;tabId=AG-PR-1069</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:269px;">2100 Bioanalyzer</td>
			<td style="width:153px;">Agilent Technologies</td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106&#38;tabId=AG-PR-1001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">PhiX Control v3</td>
			<td style="width:153px;">Illumina</td>
			<td>FC-110-3001</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.illumina.com/products/phix_control_v3.ilmn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">HiSeq 2500</td>
			<td style="width:153px;">Illumina</td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.illumina.com/systems/hiseq_2500_1500.ilmn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Pippin Prep</td>
			<td style="width:153px;">Sage Science</td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.sagescience.com/products/pippin-prep/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit 2.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32872</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.lifetechnologies.com/order/catalog/product/Q32872</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">TruSeq SR Cluster Kit v3-cBot-HS</td>
			<td>Illumina</td>
			<td>GD-401-3001</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.illumina.com/products/truseq_sr_cluster_kit_v3-cbot-hs.ilmn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TruSeq SBS Kit v3-HS</td>
			<td>Illumina</td>
			<td>FC-401-3002</td>
			<td style="width:193px;"></td>
			<td style="width:497px;">http://www.illumina.com/products/truseq_sbs_kit_v3-hs.ilmn</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">TruSeq RNA Sample prep</td>
			<td>Illumina</td>
			<td>RS-122-2001</td>
			<td style="width:193px;">Barcoded adapters used for multiplexing libraries; See Supplemental file for multiplexing protocol</td>
			<td style="width:497px;">http:/http://www.illumina.com/products/truseq-rna-access-kit.ilmn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microcentrifuge</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vortex Mixer</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dry Block Heater</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Thermal Cycler</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Water Bath</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Gel electrophoresis system</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Electrophoresis power supply</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">Gel doc</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:269px;">UV or blue light transilluminator</td>
			<td></td>
			<td></td>
			<td style="width:193px;"></td>
			<td style="width:497px;"></td>
		</tr>
	</tbody>
</table>

enhanced reduced representation bisulfite sequencing for assessment of dna methylation at base pair resolution

DNA甲基化模式的映射是大量研究在正常和病变组织。各种方法已被建立，以询问胞嘧啶甲基化模式中的细胞。全基因组测序的亚硫酸氢减少代表性的开发是为了定量检测碱基对分辨率的胞嘧啶甲基化模式在GC丰富的基因位点。这是通过结合使用一种限制性内切酶，随后通过亚硫酸氢盐转化的完成。增强精简表示亚硫酸氢盐测序（ERRBS）增加的生物相关的基因组基因座盖，并已用于分析胞嘧啶甲基化，从人，小鼠和其它生物的DNA。 ERRBS启动与DNA产生低分子量的碎片在文库制备使用限制性内切酶消化。这些片段进行标准库建设下一代测序。前的最后amplificati未甲基化的胞嘧啶的亚硫酸氢盐转化在步骤允许在有盖的基因组位点胞嘧啶甲基化水平的定量基本分辨率。该协议能在四天内完成。尽管低排序的前三个基地的复杂性，ERRBS库产生使用指定的顺序控制车道时，高质量的数据。然后，进行测绘和生物信息学分析和产率数据，可以与各种基因组范围的平台来容易地集成。 ERRBS可以利用小的输入材料数量使其可行处理人类临床样品和适用的范围内的研究应用。产生的视频演示了ERRBS协议的关键步骤。

图1提供ERRBS的概述，突出的关键步骤，这是在整个描述的协议说明。 ERRBS库用50纳克输入DNA准备。 评估准备的库的质量。图书馆例行的生产产量150-250基点和250-400基点（ 图3A-C）部分的大小。在样本库之间的尺寸分布略有差异的预期。注意，在较低和较高文库级分有非常​​激烈的DNA的大小，指示特定序列的富集。 MspI酶切位点的消化导致家庭重复DNA的序列富集存在于190基点，250基点和310基点的ERRBS库的人类基因组。这三个重复序列代表一个ERRBS库20的特性的签名（参见图3A-C和3G）。代表性的文库进行测序上的下一代序列ř采用单端读取。当在上Illumina的HiSeq 2500序推荐的库加载浓度，对500,000-700,000每平方毫米集群密度的预期。在这个聚类的聚类密度，81.6％±3.14％（N = 81），低通滤波器（ 图4A）。由于该文库插入物的低复杂度端（MspI酶切位点识别位点:C ^ CGG），测序过程中记录的强度值和质量分数，是在第3个碱基（ 图4B-C）的高度可变的，但是如果一个独立的对照泳道包括（见讨论），碱的85％将具有30或更大的质量得分（Q30值; 图4D）。 数据对准，如在协议收率碱基对分辨率的数据描述胞嘧啶甲基测定（ 表7）。人类基因组，一个51周期的单读测序的ERRBS图书馆在HiSeq 2500一条车道在高输出模式运行规律LY产生153194882±12918302共读，经过质量过滤和适配器修剪产量152231183±13189678读取输入到分析管道。平均映射效率为ERRBS库通常是62.95％±5.92％，与3183594±713547的CpG表示每10×的CpG的最小覆盖面积和每CpG的平均覆盖率84.94±16.29（N = 100）。 该ERRBS协议是经得起复用（见补充文件1:协议适配的复用测序）。从代表性的测序数据运行从复用测序运行（51周期单读测序运行总结在图5的数据; N = 128，每通道两个库; N = 11三，每个通道库; N = 11的四个库每泳道）进行比较，以一个ERRBS库的完整测序车道（51循环单阅读测序运行; N = 100），以及下采样单通道到模拟器上ê50％，33％和25％的每泳道读（2，3，和分别每泳道4样品多路复用; N = 3）。作为每个样品读取数与复用因子降低，覆盖在10倍的最小覆盖面积和每CpG的覆盖范围的CpG的数目减少，以及（ 图5和表8）。非CpG部位意味着转化率预计为99.85％±0.04％（N = 400）。转化率大于99％，降低可指示低于最佳重亚硫酸盐转化，可能导致虚假甲基化水平很高。 从具有代表性的人类基因组DNA准备了ERRBS库数据在研发2.15.2 45使用methylKit包26（参见命令的详细信息补充代码文件1）分析。该数据可以被可视化中通常使用的基因组浏览器（ 图6A）。该胞嘧啶甲基化的数据被均等地来自于两条链（ 图6B）以及范围的整频谱潜在胞嘧啶甲基化水平（ 图6C）的。 （先前描述图6E和 F和作为26）从该数据的结果（ 图6D），并涵盖在基因组基因座的广谱的CpG之间有代表性的人的DNA样品的产量高的一致性分析的技术重复。虽然技术复制品将产生高R 2的值（大于97％），生物副本将产生，R 2值范围从0.92到0.96 26，和比较不同的人类细胞类型将产生，R 2值低于0.86的（数据未示出）。 <img alt="图1" src="/files/ftp_upload/52246/52246fig1highres.jpg" /> 图1:流程图的ERRBS协议的步骤。图代表的步骤，它可以在传统的工作日内完成。*表示潜在的暂停点（立即执行<html>荷兰国际集团结扎清理和尺寸选择，协议的步骤5）之前在该样品可以在-20在进行协议的期限之前冻结℃。 <img alt="图2" src="/files/ftp_upload/52246/52246fig2highres.jpg" /> 图2:尺寸选择协议。 （A）屏幕截图在ERRBS皮平准备协议中使用的设置（见协议第5.1.2 - 5.1.6）:（1）选择纸盒类型。 （2）使用选择的标准。 （3）选择每个通道的收集模式。 （4）输入集合bp的范围。 （5）保存协议协议第5.2节中使用的人工凝胶提取（B）阶段:（1）可视化凝胶梯子。 （2）标记的尺寸为用刀片大小选择。切除标本（150-250 bp和更高的分数:250-400基点较低的分数）（3）图像。"&gt;请点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/52246/52246fig3.jpg" /> 图3:质量控制结果从使用生物分析仪机人类DNA样本准备代表ERRBS库。 （A）的凝胶状图象显示标准梯（1），低级文库级分（从皮平准备135-240碱基级分）; 2）和更高的文库级分（从皮平准备240-410碱基级分）; 3）（B）生物分析仪电泳预期低库分数（C）的预期高出库部分的生物分析仪电泳D - 。F）从质量较差库准备有代表性的数据。标准梯（1）的凝胶状图像（D）中 ，低级文库级分（2）和更高的文库级分（3）。在150bp的带标记有ARROw表示适配器过量。的下电泳（E）和更高的文库级分（F），在150碱基对（用箭头标记）过量适配器峰。一个汇集ERRBS库用于测序（G）的生物分析仪电泳。红色迹代表具有更高和更低的级分的平等代表高质量汇集库。蓝线代表一个汇集库不足以测序由于缺乏较高的和较低的分数平等代表权。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52246/52246fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52246/52246fig4highres.jpg" /> 图4:图表测序为代表ERRBS 51周期单读测序运行在HiSeq 2500测序的高输出模式。 （A）的群集的密度（K /毫米2 = 1000簇每平方毫米;蓝色）。和簇的密度通过过滤器（绿色）在两车道与ERRBS库（B）中与出现在第一30个循环中的车道的典型强度ERRBS库。请注意，从MspI酶切位点的消化前三周期的强度的CGG签名。用质量分数为30或更高（％&gt; Q30），用于在一个ERRBS车道每个周期基地（C）的百分比。（D）质量分数分布在一个ERRBS车道全部循环。蓝色=小于Q30，绿色=大于或等于Q30。在这条胡同里，基地84.7％有30或更高的质量分数。 <img alt="图5" src="/files/ftp_upload/52246/52246fig5highres.jpg" /> 图5:排序输出结果。箱形图的实验数据，从每道测序る复用和单个样本<html>纳秒（显示为绿框）从三个ERRBS库测序运行模拟下采样得出的数据和（显示为蓝色框;采样5次，每次测序运行），从51周的单读测序运行的复用系数对应每泳道ERRBS库的数目进行测序。 1 =整个车道或100％的读取和代表每通道单一ERRBS库数据; 2 = 50％车道，代表每线双ERRBS库中的数据;一泳道3 = 33％，并且表示为每泳道3 ERRBS库的数据;并且，车道4 = 25％，并代表从每泳道4 ERRBS库数据。（A）中的读出计数，或分析的序列号，每个多路复用因子。（B）中所涵盖的每个多路复用的测序数据的CpG的数因素。（C）每复用系数CpG的平均覆盖率。_blank"&gt;点击此处查看该图的放大版本。 <img alt="图6" src="/files/ftp_upload/52246/52246fig6highres.jpg" /> 图6:从人类基因组DNA准备了ERRBS库代表数据（A）美国加州大学圣克鲁兹分校（UCSC）基因组浏览器43的图像从ERRBS测序车道代表性的数据。 y轴比例尺条为0-100％甲基化，在覆盖有至少10倍的每个胞嘧啶。顶端定义轨道表示正向链及下部定制轨道表示反向链。示出的是chr12:6,489,523-6,802,422（hg19）含该基因组区域内的RefSeq基因和CpG岛（B）的分布的CpG覆盖直方图沿正向和反向链的代表性人CD34 +骨髓样品中的（C）的分布直方图的CpG甲基化水平沿两条链具有代表性的人CD34 +骨髓样品中。CpG甲基化水平从一个人的DNA样品的代表性技术复本（D）的相关性曲线图。（E）的饼图表示盖在ERRBS的CpG的比例而注释为CpG岛（浅绿色），CPG海岸（灰色）和其他地区（白色），从人类基因组DNA制成具有代表性的样本。（F）饼图说明包括在ERRBS的CpG其中注明为基因启动子的比例（红），外显子（绿色），内含子（蓝色）和间隔区（紫色）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52246/52246fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 音量 </td><td> 评论 </td></tr><tr> &lt;TD&gt; 10×T4 DNA连接酶反应缓冲液</td><td> 10微升</td><td></td></tr><tr><td>三磷酸脱氧核苷酸（的dNTP）混合的解决方案</td><td> 4微升</td><td>混合的每个核苷酸的10毫米</td></tr><tr><td> T4 DNA聚合酶</td><td> 5微升</td><td> 3000单位/ ml </td></tr><tr><td> DNA聚合酶I大（Klenow酶）片段</td><td> 1微升</td><td> 5000单位/ ml </td></tr><tr><td> T4多核苷酸激酶</td><td> 5微升</td><td>万单位/毫升</td></tr><tr><td>无DNase的水</td><td> 45微升</td><td></td></tr></tbody></table> 表1:最终修复反应的试剂的试剂名，数量用在末端修复反应（协议步骤2.1）。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 音量 </td><td> 评论 </td></tr><tr><td> 10×反应缓冲液</td><td> 5微升</td><td>例如，NEBuffer 2中加入</td></tr><tr><td> 1mM的2&#39;-脱氧腺苷5&#39;-三磷酸（的dATP） </td><td> 10微升</td><td></td></tr><tr><td> Klenow片段（3&#39;→5&#39;外切） </td><td> 3微升</td><td> 5000单位/ ml </td></tr></tbody></table> 表2:尾反应试剂，试剂的名称和数量用于尾反应（协议的步骤3.1）。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 音量 </td><td> 评论 </td></tr><tr><td>在无DNase的水为15μm退火适配器</td><td> 3微升</td><td> PE适配器1.0和PE适配器2.0; 见表4序列和参考</td></tr><tr><td> 10×T4 DNA连接酶反应缓冲液</td><td>五81，L </td><td></td></tr><tr><td> T4 DNA连接酶</td><td> 1微升</td><td> 200万单位/毫升</td></tr><tr><td>无DNase的水</td><td> 31微升</td><td></td></tr></tbody></table> 表3:适配器连接反应试剂的试剂名，并在适配器连接反应（协议步骤4.2）所使用的量。 <img alt="表4" src="/files/ftp_upload/52246/52246table4.jpg" width="700" /> 表4:在整个连接反应（协议步骤4）和PCR扩增步骤（协议步骤7）ERRBS协议中使用的寡核苷酸在ERRBS协议中使用寡核苷酸的列表。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 音量 </td><td> 评论 </td></tr><tr><td> 10倍的FastStart高保真反应缓冲液的Wi第18毫氯化镁</td><td> 20微升</td><td></td></tr><tr><td> 10毫米的dNTP混合解决方案</td><td> 5微升</td><td></td></tr><tr><td> 25μMPCR PE底漆1.0 </td><td> 4微升</td><td> 见表4 </td></tr><tr><td> 25μMPCR PE底漆2.0 </td><td> 4微升</td><td> 见表4 </td></tr><tr><td>的FastStart高保真酶</td><td> 2微升</td><td> 5单位/微升的FastStart Taq DNA聚合酶</td></tr><tr><td>无DNase的水</td><td> 125微升</td><td></td></tr></tbody></table> 表5:PCR反应试剂的试剂名，并在PCR扩增反应（协议步骤7.1）所使用的量。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td rowspan="2"> 协议的步骤 </td><td rowspan="2"> 试剂/协议细节 </stron克&gt; </td><td colspan="3"> 输入DNA量 </td></tr><tr><td> 5-10纳克</td><td> 25纳克</td><td> 50纳克</td></tr><tr><td rowspan="2"> 1 </td><td> MspI酶切位点的酶</td><td> 1微升</td><td> 2微升</td><td> 2微升</td></tr><tr><td> MspI酶切位点消化反应体积</td><td> 50 </td><td> 100 </td><td> 100 </td></tr><tr><td rowspan="2"> 4 </td><td>在连接反应适配器</td><td> 1微升</td><td> 2微升</td><td> 3微升</td></tr><tr><td>连接反应体积</td><td> 20微升</td><td> 25微升</td><td> 50微升</td></tr><tr><td>五</td><td>大小选择协议</td><td>只有手动胶</td><td>皮蓬准备或手动胶</td><td>皮蓬准备或手动胶</td></tr><tr><td rowspan="2"> 7 </td><td> PCR引物浓度</td><td> 25μM </td><td> 25μM </td><td> 10μM的14个循环; 25μM18次</td></TR&gt; <tr><td> PCR循环数</td><td> 18 </td><td> 18 </td><td> 14-18 </td></tr></tbody></table> 表6:协议步骤修改为输入材料数量范围从5-50纳克几个步骤在整个协议要求用来产生从不同数量的起始材料的高品质的文库试剂量的修饰。被更改的关键试剂的量包括在这里。调节在反应缓冲液和水体积相应。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> CHR </td><td> 基地 </td><td> 缕 </td><td> 复盖 </td><td> freqC </td><td> freqT </td></tr><tr><td> CHR 1 </td><td> 10564 </td><td> ř </td><td> 366 </td><td> 85.52 </td><td> 14.48 </td></tr><tr><td> CHR 1 </td><td> 10571 </td><td>˚F </td><td> 423 </td><td> 91.25 </td><td> 8.75 </td></tr><tr><td> CHR 1 </td><td> 10542 </td><td> ˚F </td><td> 432 </td><td> 91.2 </td><td> 8.8 </td></tr><tr><td> CHR 1 </td><td> 10563 </td><td> ˚F </td><td> 429 </td><td> 94.64 </td><td> 5.36 </td></tr><tr><td> CHR 1 </td><td> 10572 </td><td> ř </td><td> 366 </td><td> 96.99 </td><td> 3.01 </td></tr><tr><td> CHR 1 </td><td> 10590 </td><td> ř </td><td> 370 </td><td> 88.11 </td><td> 11.89 </td></tr><tr><td> CHR 1 </td><td> 10526 </td><td> ř </td><td> 350 </td><td> 92 </td><td> 8 </td></tr><tr><td> CHR 1 </td><td> 10543 </td><td> ř </td><td> 368 </td><td> 92.93 </td><td> 7.07 </td></tr><tr><td> CHR 1 </td><td> 10525 </td><td> ˚F </td><td> 433 </td><td> 91.92 </td><td> 8.08 </td></tr><tr><td> CHR 1 </td><td> 10497 </td><td> ˚F </td><td> 435 </td><td> 88.74 </td><td> 11.26 </td></tr></tbody></table> &lt;P类="jove_content"&gt; 表7:代表ERRBS数据。获得碱基对数据的数据对齐和胞嘧啶甲基化的决心，经过对每个CpG的覆盖，对齐协议描述将决定基因组坐标（列:字符=染色体，基地和钢绞线），具体的位点的覆盖率（覆盖），以及检测胞嘧啶与胸苷百分数（freqC和freqT的分别率）。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 每通道ERRBS库数量 </td><td> 平均数的唯一对准读取 </td><td> 平均覆盖的CpG数量 </td><td> 平均每CpG的覆盖范围 </td></tr><tr><td> 1 </td><td> 152231184±13189678 </td><td> 3183594±713547 </td><td> 85±16 </td></tr><tr><td> 2 </td><td> 77680837±7657058 </td><td> 2674823±153494 </td><td> 49±9 </td></tr><tr><td> 3 </td><td> 49938156±2436865 </td><td> 2552186± - 76624 </td><td> 39±2 </td></tr><tr><td> 4 </td><td> 34457208±4441686 </td><td> 1814461±144339 </td><td> 28±4 </td></tr></tbody></table> 表8:从单一的测序和多路复用ERRBS图书馆代表的参数显示的是每个通道的数据，从51周的单读测序运行:均值和独特的排列标准偏差读，覆盖CpG岛的数量和覆盖面从每单测序获得的CpG位点每通道ERRBS库（N = 100），每通道2 ERRBS库（N = 128），每通道（N = 11）三种ERRBS库和每通道（N = 11）4 ERRBS库。

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Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

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25.4K 次观看.  Weill Cornell Medical College. Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

视频: Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

科研

生物学

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA甲基化:亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If humans are to travel beyond low Earth orbit, a transition towards space-ready, point-of-care (POC) testing is required. Such testing needs to be comprehensive, easy to perform in a reduced-gravity environment, and unaffected by the stresses of launch and spaceflight. Countless POC devices have been developed to mimic laboratory scale counterparts, but most have narrow applications and few have demonstrable use in an in-flight, reduced-gravity environment. In fact, demonstrations of biomedical diagnostics in reduced gravity are limited altogether, making component choice and certain logistical challenges difficult to approach when seeking to test new technology. To help fill the void, we are presenting a modular method for the construction and operation of a prototype blood diagnostic device and its associated parabolic flight test rig that meet the standards for flight-testing onboard a parabolic flight, reduced-gravity aircraft. The method first focuses on rig assembly for in-flight, reduced-gravity testing of a flow cytometer and a companion microfluidic mixing chip. Components are adaptable to other designs and some custom components, such as a microvolume sample loader and the micromixer may be of particular interest. The method then shifts focus to flight preparation, by offering guidelines and suggestions to prepare for a successful flight test with regard to user training, development of a standard operating procedure (SOP), and other issues. Finally, in-flight experimental procedures specific to our demonstrations are described.

Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

样机小型化流式细胞仪及配套的混合微流控技术降低重力环境硬件演示

Until recently, astronaut blood samples were collected in-flight, transported to earth on the Space Shuttle, and analyzed in terrestrial laboratories. If ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

在体外甲基化分析研究蛋白质精氨酸甲基化

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

关联特定基因的DNA甲基化与表达的变化和星形胶质细胞的转录活性<em&gt; KCNJ10</em&gt;（Kir4.1）

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...