我们感谢奥里亚瓦迪对生成的数字援助。在IS组的工作是由以色列科学基金会（批准号:十分之五百六十七）和欧洲研究理事会资助启动（＃281306）的支持。

注意:职责范围只能上生长，不同步的细胞来测量。 2×10 6个快速生长的细胞，这通常会导致在〜1×10 5个细胞在S期（速率限制步骤） -一个程序应当至少有1开始。它建议进行使用两个或三个重复各实验。 CNR-TOR的整个过程可以在一个星期内完成 - 两个天应专用于所有步骤达文库制备，需要用于测序一至两天，另外天是必要的初始数据分析。 1.培养细胞的采集注:该协议是为于10cm板（含有约2 - 5×10 6个细胞）中培养生长的细胞编写的，但可以很容易地调整到其他平台。 <ol><li>对于悬浮生长的细胞，进行固定（第2节）。 </li><li>对于贴壁细胞，吸洗了PL用3毫升的PBS吃不含Ca 2+和镁离子 。 </li><li>丢弃PBS中，并用1mL商业胰蛋白酶-EDTA孵育细胞5分钟，在37℃下直到细胞分离。 注:胰蛋白酶处理的持续时间应调整到不同的细胞类型。 </li><li>加入3毫升培养基中和胰蛋白酶，并收集在15毫升锥形管或一个5毫升聚苯乙烯管细胞。置于冰上。 </li></ol> 2.固定注:对于这部分，所有的步骤应在4℃下进行。 <ol><li>离心细胞，在300×g离心在4℃下5分钟。 </li><li>洗板细胞两次用1毫升冷PBS。 </li><li>悬浮细胞在250微升（总）冷PBS。 </li><li>同时轻轻涡旋管，慢慢°C 100％的乙醇添加-20滴加800微升。这导致70的最终乙醇浓度 - 80％。 注意:高纯度乙醇建议在此步骤。 </li><li>孵育细胞在冰上FO-R 30分钟。 注:在此阶段细胞可以保持几天在4℃或在-20℃下几个月。 </li></ol> 3. PI染色<ol><li>离心细胞，在500×g离心在4℃下10分钟。 </li><li>小心吸出上清液并用1mL冷PBS洗涤细胞两次。 </li><li>吸液和悬浮用下列混合物各样品:1毫升的PBS，5微升10毫克/毫升RNA酶A，50微升1mg / mL的碘化丙啶（PI;使用前混合瓶）。调整浓度可达〜2×10 6个细胞/ mL）。 注意:远离光 - PI对光敏感。 </li><li>通过35微米网状到5毫升聚苯乙烯管过滤器，并关闭封口膜。 </li><li>孵育在室温在黑暗中，15 - 30分钟。 注:细胞现在已经准备好FACS分析。如果需要，染色的细胞可以在4℃保存至少24小时在黑暗中。 </li></ol> 4.排序<ol><li>使用FACS机器排序细胞。我们e为561纳米的激光来区分基于其PI强度细胞。靠近535 nm激发最大像488纳米或532其它激光器可以根据FACS机器的配置中使用。 </li><li>为获得最佳结果，使用推荐用于特定小区的大小（对于大多数细胞85微米）最小的喷嘴。优先于产量纯度模式。使用稳定的缓慢流动，通常高达300-500事件/秒，以45 psi的鞘压力。 </li><li>使用选通，通过绘制FCS VS SSC区分死细胞和亚细胞碎片（低FCS和高SSC）。从活细胞通过绘制SSC-宽度（W） 与后跟一个FSC-W VS FSC-H曲线图，并通过PI-W¯¯SSC-高度（H）的区别双峰VS的PI-H（双双将具有相同的H值但更大的W值）。的活单细胞绘制其表示细胞的DNA含量的PI-区（A）的强度的直方图。 </li><li>排序细胞进入G1和S期， 如图1。门控对于s应宽和侵入G1和G2期，而G1门应该是狭窄，尽量选自S越好。 </li></ol><img alt="图1" src="/files/ftp_upload/55157/55157fig1.jpg" /> 图1.细胞周期的阶段决定基于PI的强度。柱状图表示细胞DNA含量的小鼠胚胎成纤维细胞（MEF）总体的分布（由PI-区测量）。 DNA含量用于人口排序成两个亚群ⅰ）G1期细胞（2N DNA含量）和ii）S期细胞（2N - 4N DNA含量），使用标记的区域。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55157/55157fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 注:G1期细胞的集合的目的是考虑到在不同的基因组区域之间的测序效率偏差。另一种应用蟑螂是使用G1停滞细胞来自相同细胞类型。这种方法使清洁剂的结果（因为它最大限度地减少S期的污染），但它可能会引入偏见从被捕细胞和细胞测量之间的遗传差异所产生的。 <ol start="5"><li>排序在寒冷条件下，收集排序细胞到1.5 / 2 mL管。保持排序在冰上管。 注意:为了提高DNA回收率，最好是使用低的结合管或使用涂有4个试管-在4℃下18 3小时- 5％BSA的1。 </li></ol> 5. DNA纯化<ol><li>对于每个样本（G1和S）用DNA纯化试剂盒纯化DNA。 注:当使用市售的试剂盒，洗脱用400微升的洗脱缓冲液到2mL管产量高，如制造商推荐的。 </li><li>使用荧光检查DNA浓度。 注:从由FACS收集100000哺乳动物细胞中，人们应该得到的DNA〜1微克。一个吨该阶段DNA可在4℃或在-20℃长期储存保持几天。 </li></ol> 6.超声<ol><li>转移DNA导入1.7毫升管，其是与所使用的磁性底座兼容。 </li><li>集中的DNA根据制造商的说明使用2倍SPRI珠，用磁性支架，并在50微升洗脱缓冲液洗脱。 </li><li>剪切DNA用聚焦超声发生器到250基点的平均目标峰大小。使用以下设置为50μLDNA样本:50 W，20％占空比系数，每爆200个周期，20℃，120秒。 </li><li>验证通过电泳的剪切的DNA的大小。推荐大小分布为200 - 700个碱基，在〜250 bp的高峰。 注:在此阶段的DNA可以保持几天，在4℃或在-20℃长期保存。 </li></ol> 7.文库制备和测序注意:许多文库制备试剂盒和不同耳鼻喉科测序平台应的工作方式类似于我们所使用，并提到在材料部分的人。实际上，在过去，职责范围的地图用与微阵列平台2非常相似的方法产生的。 <ol><li>准备使用任何商业库制备试剂盒库。 </li><li> 800 bp的 - 在图书馆准备结束时，利用磁珠300选择大小。 </li><li>准备库后，使用荧光测定DNA浓度。 </li><li>使用电泳测量DNA大小。 </li><li>在任何平台上进行测序。 注意:每个样品测序至少10μM的读取建议。该深度相当于一个读大约每300bp的（为〜3千兆大小的基因组），并足以为Tor测量在50，分辨率 - 100 kb的。增加深度将导致在窗口的尺寸的减小，从而使增加与更高的确定性的分辨率。配对末端测序并非必要因为只有覆盖信息这个协议被收集。然而，它可能会帮助解决的位置读取包含重复序列。 </li></ol> 8.分析注意:数据分析是基于由A.科伦等人所使用的方法。 19。 <ol><li>地图测序数据使用bowtie2或任何可靠的短读取对准相应的基因组。定义不同的尺寸，相当于覆盖染色体窗口由200段涵盖在G1分数读取和计数S期读取同一个窗口。 </li><li>计算每个窗口在S / G1比。这应该产生与沿基因组中（ 图3）在S / G1比大的波动的地图。良好控制职责范围测量的可靠性是在该地图进行比较，以在G1 / G1比（从G1的两个独立测量值），其应该是更平坦。 </li><li>数据标准化为0均值和1个SD通过各V减去ALUE所有窗口（不包括X染色体）和由所有窗口的S / G1的标准偏差除以结果的平均值。这样做是为了将其转换为z值和使不同实验之间的比较完成的。 </li><li>除去由UCSC基因组浏览器以及包含少于15个数据窗每个剩余间间隙片段列出的所有的间隙区域。 </li><li>通过与10个参数的MATLAB函数csaps一个三次样条函数平滑剩余的碎片- 16，并在设定点插每100 KB。 注:平滑和内插的参数应该根据数据的深度来调节。其它合适的平滑化方法和功能存在，并且可以使用。 </li><li>视觉上确认的每个复制的可靠性后，合并所有的读取和通过对这个数据与上述相同的过程计算更深分辨率轮廓。 </li></ol>

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No conflicts of interest declared.

CNR-TOR可以在原则上任何真核增殖细胞群，可以通过FACS对S和G1期（由莱因德N。和吉尔伯特DM 20中综述）被划分来执行。这里所描述的方法已被调整到哺乳动物细胞用〜3千兆的基因组大小如人和小鼠。在CNR-TOR协议的微小变化（在细胞制剂和测序深度）都需要，以便将其调整到其它真核生物。因为它是限速步骤必须注意到的足够量的S期的细胞的集合。因此，对于细胞周期的初步FACS应执行，以确认该实验可以做到，并验证在S期的细胞的量。为表现出不规则的细胞周期的细胞，它是建议prestain细胞用BrdU检测分裂的细胞，并按照抗BrdU制造商协议，直至FACS步骤。通常细胞用10-20微米的BrdU 30分钟。 <p类="jove_content"&gt;建议以执行生物一式三份每个实验，以检测可能由在细胞生长和在分类处理的差异干不一致。这是为的细胞类型和条件之间的职责范围的地图的微小变化的识别特别重要。对于快速生长的细胞，建议使用单一10厘米组织培养板/烧瓶（含有约2-5×10 6细胞）的每个中，以获得&gt; 1×10 5个细胞在S期复制。该量应与缓慢生长的细胞时可以增加，因为细胞在S期的百分比较低，因此人们必须更多的细胞，以获得在S细胞的足够量的排序当排序单元，它根据它们的DNA含量，因此，建议使用低流速来实现细胞的可能的最佳分离是很重要的。为了最大限度地提高时间分辨率，它以收集细胞是重要从整个S期。这是通过为宽选通能够像可能的（ 图1）来实现的。另一方面，G1浇注应窄（从G1峰和向左开始; 图1），以避免两个子G1和早S期污染。 超声处理可通过各种方法来进行，但它是推荐使用的聚焦超声发生器使一个强大的和容易超声处理，而不需要为每个实验的校准。然而，首先建立在实验室的协议时，建议校准参数用于获得200-700碱基对的尺寸分布。 正如介绍中解释的，也有全基因组的职责范围映射两种主要的方法 - 的BrdU-IP和CNR-TOR。这两种方法都得到尽管它们之间的差异相似职责范围的地图（数据未示出）。因为最大的区别betwe的CNR-TOR方法是在铀浓缩范围的限制恩早期和晚期的地区是双重的，而用的BrdU-IP更高的富集实现，年初以来复制的区域将在早期几乎分数只包含BrdU的。另一方面，所述的BrdU-IP方法分辨率由收集S期级分的数量的限制。在其最常见的应用中，只有两个级分（早期与晚期S）相比较，这导致了细时间分辨率的一个折衷。的CNR-TOR方法，然而，给出了沿整个S期的连续信号。此外，BrdU的IP的方法是基于免疫沉淀通常给出了一个比CNR-TOR方法高得多的背景。的CNR-TOR方法的另一个优点是，它可被追溯用于提取从非同步培养的测序数据职责范围信息作为最近描述21。最后，CNR-TOR方法优选由于其简单性和由于这样的事实，它可以因为它缩小的不是基于IMMU无沉淀。 职责范围的基因组和表观特征的复杂网络中的作用仍然被破译。相对轻松CNR-TOR法允许在现有职责范围数据的扩展，包括了许多的自然条件使细胞的经验和应彻底探索和基因组范围。这包括各种复制压力的情况下，核内复制以及各种癌症变换。利用SNP数据和更高的测序深度也将允许单独测量每个等位基因的职责范围。此外，在测序成本进一步降低将使允许的条件之间的细微变化的识别职责范围的地图的分辨率，这可以进一步有助于增加。其他未来的应用可以通过改善现有的方法和细胞，使体内的测量职责范围小的数字将职责范围的测量来实现。

哺乳动物DNA复制是严格调节细胞周期过程中，以确保精确地一次每个染色体的精确复制。复制根据一个高度管制的次序发生-多个大型基因组区域（〜兆）S期的开始复制（早期复制域），而其他的基因组区域的中后期S期（中晚期复制域）后重复1。大多数基因组的复制在所有组织中（组成职责范围域）的同时，而30％ -在基因组的50％，也期间癌症转化5改变组织2之间它的职责范围，分化3,4期间和在较小程度上。此外，某些基因组区域复制异步6，7，8，即是有区别的在两个等位基因之间的职责范围。 职责范围与许多基因组和表观基因的功能，包括转录水平，GC含量，染色质的状态，基因密度等 1,9相关。职责范围也与突变率和类型10，11，因此毫不奇怪相关联，则复制程序的扰动与癌症12,13。职责范围和染色质结构之间的因果关系尚不清楚。这可能是开放的染色质促进早期复制。然而，另一种模型表明，在复制过程中的染色质组装和存在于开头的不同染色质调节和S期引线端至差分早期和晚期复制区域1的包装，14 </sup&gt;。我们最近表明，职责范围形状通过影响发生在不同的基因组区域11突变型GC含量。 荧光原位杂交（FISH）是在各个基因检测职责范围的主要方法。它是通过计算显示出单一的FISH信号与双重对于给定的等位基因15,16百分比S期细胞的百分比简单地进行。一种替代方法，由脉冲的标记用BrdU的DNA，根据它们的DNA含量，以多个时间点沿小号分拣细胞，免疫沉淀含有BrdU的DNA，并检查沉淀的DNA的丰度与定量PCR 17。 基因组职责范围映射可以通过两种方法来实现。第一种方法是上述的的BrdU-IP为基础的方法的基因组的版本，其中，所述量的量化在每个级分沉淀的DNA被用于通过杂交整个基因组，以微阵列或通过深度测序同时进行。第二种方法，CNR-TOR，基于由在G1期细胞的DNA含量测定S期细胞和正火的每个基因组区域的拷贝数。在该方法中，细胞通过FACS分类成非复制（G1期），并复制（S期）基团（ 图1）。在G1期细胞在所有的基因组区域的相同拷贝数和因此它们的DNA含量应该是相同的。另一方面，在S中的DNA拷贝数依赖于职责范围，因为早期复制区域中大多数细胞进行复制，因此，它们的DNA含量增加一倍，而晚复制区域没有在大多数细胞尚未复制，因此，它们的DNA含量将是类似的G1期细胞。因此，S键DNA含量的G1比例指示职责范围。 DNA的每个基因组区域的量测得的通过杂交要么芯片或深度测序2,8。的CNR-TOR方法的优点将被进一步讨论。 本文介绍在图2描述了基因组职责范围映射CNR-TOR方法。本文从收集细胞，直到结果的基本分析和基因组职责范围的地图的创建讨论的全过程的细节。在本文中描述的协议已经在培养中生长的各种细胞类型被成功执行。这个协议的未来的改进可导致职责范围体内的映射和在罕见的细胞类型。

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">PBS</td>
			<td style="width:157px;">BI (Biological Industries)</td>
			<td style="width:119px;">02-023-1A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA</td>
			<td>BI (Biological Industries)</td>
			<td>03-052-1B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15ml conical tube</td>
			<td>Corning</td>
			<td align="right">430790</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5ml Polystyrene round Bottom tube with cell strainer cap&#160;</td>
			<td>BD-Falcon</td>
			<td align="right">352235</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Gadot</td>
			<td>64-17-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse-A 10mg/ml</td>
			<td>Sigma</td>
			<td>R4875</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidiom iodide 1mg/ml</td>
			<td>Sigma</td>
			<td>P4170</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">parafilm</td>
			<td>Parafilm</td>
			<td>PM-996</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5ml DNA LoBind Eppendorf tubes&#160;</td>
			<td>Eppendorf</td>
			<td align="right">22431021</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7ml MaxyClear tube&#160;</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">magnetic beads - Agencourt AMPure XP&#160;</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasonicator</td>
			<td>Covaris</td>
			<td>M-series&#160; -530092</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 &#181;l microTUBE AFA Fiber Screw-Cap 6x16mm</td>
			<td>Covaris</td>
			<td align="right">520096</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit fluorometer</td>
			<td>Invitrogen</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Qubit dsDNA High Sensitivity (HS) Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32854</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrophoresis.2200 Tape station system</td>
			<td>Agilent</td>
			<td>D1000 ScreenTape</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Seqeuncing - Illumina NextSeq system</td>
			<td>Illumina</td>
			<td>SY-415-1001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dneasy kit for DNA purification</td>
			<td>Qiagen</td>
			<td align="right">69504</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureProteom Magnetic Stand</td>
			<td>Millipore</td>
			<td>LSKMAGS08</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-BrdU/FITC</td>
			<td>DAKO</td>
			<td>F7210</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS sorter</td>
			<td>BD</td>
			<td>FACSARIA III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS software</td>
			<td>BD</td>
			<td>FACSDiva v 8.0.1</td>
		</tr>
	</tbody>
</table>

genome-wide determination of mammalian replication timing by dna content measurement

基因组的复制过程中在一个高度调节的过程，以确保DNA复制的保真度在细胞周期的S期时发生。每个基因组区域是在不同的时间在S期通过复制多个起源的同时激活复制。复制（TOR）的时间与许多基因组和表观遗传特征相关，并链接到突变率和癌症。解读复制程序的完整基因组认为，在健康和疾病是未来的一个主要目标和挑战。 本文详细介绍了该法的"S / G1的用于映射复制基因组时代拷贝数比率"（以下称为:CNR-TOR），一个简单的方法来绘制哺乳动物细胞的基因组范围的职责范围。该方法是基于S期细胞和G1期细胞的拷贝数的差异。在6个步骤进行CNR-TOR方法:1，细胞和染色与碘化丙啶（PI）的制备; 2.索尔廷G1和使用荧光激活细胞分选S期细胞（FACS）; 3. DNA纯化; 4.超声; 5.文库制备和测序; （六）生物信息学分析。该CNR-TOR法是一种快速简便的办法，导致了详细的复制地图。

一个典型的职责范围的地图显示在图3为小鼠胚胎成纤维细胞（MEFs）。该图演示了分析过程，因为它同时显示了点，这是各个窗口（步骤8.3）的归一化的S / G1期的比例，以及从该立方平滑和内插（步骤8.5）产生的线。 这类地图捕获复制程序的组织，这是两种类型的职责范围的域的拼凑:ⅰ）大的区域（在一个兆碱基的顺序），其同时在早期复制（点击率=恒定职责范围区域），中间或晚期小号;及ii）暂时过渡区域（TTRS），其中职责范围逐渐变化。点击率通过TTRS彼此连接并一起这两种类型的复制机构的覆盖整个基因组。 尽管相对较低的测序Ç<html>超龄（一个读每300个基点），用于职责范围测绘，由此产生的地图是相当强劲。 图4示出了职责范围的地图相比，在同一区域鼠前B细胞的一式三份的MEF细胞的一式三份的再现性。这类地图之间的比较允许其被定义为在其中组织之间在职责范围的地图的差异比重复之间的差异显著较大区域差分职责范围的区域的鉴定。 <img alt="图2" src="/files/ftp_upload/55157/55157fig2.jpg" /> 图2:协议方案。示意图描述CNR-TOR方法的步骤。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55157/55157fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 57 / 55157fig3.jpg"/&gt; 图3:代表基因组职责范围的地图。显示的是MEF细胞的染色体全1的职权范围，并在〜50 MB区一探究竟。示出的是在S / G1值的变化的大小的窗口（点）和平滑化数据（实线）的Z值。高S / G1值对应于早期的复制，而较低的值对应于晚复制。箭头指向恒定职责范围区域的例子（点击率;红色）和时间的过渡区（TTRS;绿色）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55157/55157fig3large.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/55157/55157fig4.jpg" /> 图4:重复再现性。相比于前B一式三份（红色）在18 MB区域的鼠标1号染色体橙色箭头平滑MEF一式三份的职责范围（蓝色）（A）指向细胞系之间差的区域的例子。 （B）中的6个样品之间的Spearman相关性的热图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55157/55157fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement at JoVE.com

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

genome-wide-determination-mammalian-replication-timing-dna-content

Nanyang Technological University

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JoVE Journal

Genetics

8.4K 次观看.  Hebrew University of Jerusalem. We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

视频: Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

在 果蝇卵巢成人线粒体DNA复制的原位标记埃杜染色

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

科研

遗传学

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

滞后链DNA合成的动力学研究<em&gt;体外</em&gt;由T7噬菌体复制蛋白

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

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

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

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

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

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

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

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

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

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

ATAC 基因组在原发性人类 T 淋巴细胞中的染色体全易接近染色质

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

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

增殖和静止人成纤维细胞的全基因组转录衰变率的测定

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

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

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

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

启动子捕获高分辨率, 全基因组的启动子交互分析

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

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...