这项工作得到了葛兰研究院 GM117446 的支持。

1. 流式细胞仪分选细胞的制备
<ol><li>接种15毫升试管, 每 YEPD 汤8毫升, 这样, 在隔夜生长后, 每毫升的培养量达到 5 x 106到 1.5 x 107细胞的密度 (见讨论说明 1)。</li>
	<li>向下旋转酵母细胞 (1400 x g, 在室温或4°c) 在15毫升螺丝帽离心机管为5分钟, 并用重悬细胞在1.5 毫升70% 乙醇, 并且转移到1.6 毫升离心管, 让这个坐 1 h 在室温或至少 3 h 在冰 (可以无限期地存储在4摄氏度) (参见讨论点 (2))。</li>
	<li>在离心的酵母细胞 (1.4万 x g, 4 °c) 为1分钟, 并用重悬在1毫升50毫米柠檬酸钠, pH 7.2, 旋转下来 (1.4万 x g, 4 °c) 为1分钟, 吸出上清, 并用重悬在1毫升50毫米柠檬酸钠, pH 7。2, 含有0.25 毫克/毫升 RNAse 溶液, 1 小时在50摄氏度或隔夜在37°c 在热块或水浴。</li>
	<li>添加50µL 蛋白酶 K 溶液 (20 毫克/毫升悬浮10毫米三 pH 7.5, 2 毫米 CaCl2, 50% 重到容量甘油) 和孵育 1 h 在50°c。</li>
	<li>自旋下细胞 (1.4万 x g, 4 °c), 并用重悬细胞在1毫升50毫米柠檬酸钠, pH 值 7.2, 旋转下来 (1.4万 x g, 4 °c), 吸出上清与真空, 并用重悬在1毫升1µM 绿色核酸菌株悬浮在50毫米枸橼酸钠, pH 值 7.2, 在黑暗中孵育1小时室温, 油脂实验 2x, 每2秒 (输出功率2瓦)。</li>
</ol>2. 细胞分类
<ol><li>使用细胞分拣器, 根据 DNA 含量将细胞分类为 G1、S、"早期 G2" 和 "晚期 G2", 从每个细胞周期阶段收集至少160万个单倍体细胞, 如图 1 (参见讨论点 (3))。</li>
	<li>将细胞转移到离心管, 在 1.4万 x g 离心4摄氏度时, 将细胞旋转20分钟, 并吸进上清, 真空 (颗粒可无限期贮存在-20 °c 处; 参见讨论点 (4))。</li>
</ol>3. DNA 提取和测序库的编制
注: 以下步骤是基于 "协议 I" 在酵母基因组 DNA 提取套件 (见材料表)。
<ol><li>添加120µL 的 "消化缓冲", 一个专有的缓冲, 允许酵母溶解酶消化细胞壁, 5 µL 酵母裂解酶, 并用重悬涡流, 孵化37°c 40-60 分钟。</li>
	<li>添加120µL 的 "裂解缓冲", 一个专有的缓冲, 其中包含洗涤剂, 和漩涡硬的10二十年代。</li>
	<li>添加250µL 氯仿-混合彻底1分钟。</li>
	<li>在离心中以最大速度旋转2分钟。</li>
	<li>将上清液载入 DNA 结合柱上, 离心机以最大速度在离心1分钟。</li>
	<li>添加300µL "DNA 洗涤缓冲器", 一个专有的缓冲, 其中含有乙醇, 和离心机1分钟的最大速度在一个离心。丢弃流量, 添加300µL 的 DNA 洗涤缓冲液, 重复洗涤过程。</li>
	<li>将旋转柱转移到新鲜的离心管, 添加60µL (不 TE)-等待 1-3 分钟, 然后离心机十年代洗脱的 DNA。</li>
	<li>测量浓度通过添加5µL 的 DNA 到195µL 的 dsDNA 高灵敏度试剂已稀释 1:200 dsDNA 高灵敏度缓冲, 然后测量荧光荧光计, 使用10µL 的提供标准校准;预计10和50的 DNA 总产量。</li>
	<li>油脂实验 (峰值事件功率 450, 值班系数 30%, 周期每突发 200, 治疗时间六十年代, 水位 6), 以打破 DNA 成 250-350 bp 碎片。</li>
	<li>使用 dna 样本准备套件和序列 (至少 1000万 50 bp 单端读取) 在快速模式下的 dna 测序仪上准备一个库 (参见讨论说明 (5))。</li>
</ol>4. 对序列数据的分析和复制配置文件的生成
<ol><li>使用 Gsnap12将序列与酿酒酵母sacCer3 基因组对齐 (请参阅讨论说明 (6))。</li>
	<li>使用 Bedtools "genomeCoverageBed" 软件13确定每基对读取深度。</li>
	<li>删除读深度中的 artefactual 峰值 (如果存在) (请参见讨论说明 (7))。</li>
	<li>用中线在 20 kb 滑动窗口中使用 "runMean" 函数在 R 包 "caTools" 中, 通过染色体平滑读取深度 (参见讨论说明 (8))。</li>
	<li>将 S 阶段、早期 G2 和后期 G2 的读取深度绘制为完全复制的读取深度的百分比 (请参见讨论说明 (9))。</li>
</ol>

<ol><li>Fragkos, M., Ganier, O., Coulombe, P., Mechali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+replication+origin+activation+in+space+and+time%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">DNA replication origin activation in space and time.</a> Nat Rev Mol Cell Biol. 16, 360-374 (2015).</li>
<li>Santocanale, C., Diffley, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Mec1-+and+Rad53-dependent+checkpoint+controls+late-firing+origins+of+DNA+replication%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A Mec1- and Rad53-dependent checkpoint controls late-firing origins of DNA replication.</a> Nature. 395, 615-618 (1998).</li>
<li>Brewer, B. J., Fangman, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+replication+fork+barrier+at+the+3%27+end+of+yeast+ribosomal+RNA+genes%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A replication fork barrier at the 3' end of yeast ribosomal RNA genes.</a> Cell. 55, 637-643 (1988).</li>
<li>Raghuraman, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replication+dynamics+of+the+yeast+genome%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Replication dynamics of the yeast genome.</a> Science. 294, 115-121 (2001).</li>
<li>Muller, C. A., Nieduszynski, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conservation+of+replication+timing+reveals+global+and+local+regulation+of+replication+origin+activity%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Conservation of replication timing reveals global and local regulation of replication origin activity.</a> Genome Res. 22, 1953-1962 (2012).</li>
<li>Koren, A., Soifer, I., Barkai, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MRC1-dependent+scaling+of+the+budding+yeast+DNA+replication+timing+program%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">MRC1-dependent scaling of the budding yeast DNA replication timing program.</a> Genome Res. 20, 781-790 (2010).</li>
<li>Lang, G. I., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutation+rates+across+budding+yeast+chromosome+VI+are+correlated+with+replication+timing%5BTitle%5D)+AND+%22Genome+Biol+Evol%22%5BJournal%5D)">Mutation rates across budding yeast chromosome VI are correlated with replication timing.</a> Genome Biol Evol. 3, 799-811 (2011).</li>
<li>Durkin, S. G., Glover, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome+fragile+sites%5BTitle%5D)+AND+%22Annu+Rev+Genet%22%5BJournal%5D)">Chromosome fragile sites.</a> Annu Rev Genet. 41, 169-192 (2007).</li>
<li>Dillon, L. W., Burrow, A. A., Wang, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+instability+at+chromosomal+fragile+sites+in+cancer%5BTitle%5D)+AND+%22Curr+Genomics%22%5BJournal%5D)">DNA instability at chromosomal fragile sites in cancer.</a> Curr Genomics. 11, 326-337 (2010).</li>
<li>Widrow, R. J., Hansen, R. S., Kawame, H., Gartler, S. M., Laird, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Very+late+DNA+replication+in+the+human+cell+cycle%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Very late DNA replication in the human cell cycle.</a> Proc Natl Acad Sci U S A. 95, 11246-11250 (1998).</li>
<li>Foss, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SIR2+suppresses+replication+gaps+and+genome+instability+by+balancing+replication+between+repetitive+and+unique+sequences%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">SIR2 suppresses replication gaps and genome instability by balancing replication between repetitive and unique sequences.</a> Proc Natl Acad Sci U S A. 114, 552-557 (2017).</li>
<li>Wu, T. D., Reeder, J., Lawrence, M., Becker, G., Brauer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GMAP+and+GSNAP+for+Genomic+Sequence+Alignment%3A+Enhancements+to+Speed%2C+Accuracy%2C+and+Functionality%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">GMAP and GSNAP for Genomic Sequence Alignment: Enhancements to Speed, Accuracy, and Functionality.</a> Methods Mol Biol. 1418, 283-334 (2016).</li>
<li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BEDTools%3A+a+flexible+suite+of+utilities+for+comparing+genomic+features%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26, 841-842 (2010).</li>
<li>Langmead, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aligning+short+sequencing+reads+with+Bowtie%5BTitle%5D)+AND+%22Curr+Protoc+Bioinformatics%22%5BJournal%5D)">Aligning short sequencing reads with Bowtie.</a> Curr Protoc Bioinformatics. , Chapter 11, Unit 11 17(2010).</li>
<li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25, 1754-1760 (2009).</li>
<li>Kellis, M., Birren, B. W., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proof+and+evolutionary+analysis+of+ancient+genome+duplication+in+the+yeast+Saccharomyces+cerevisiae%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae.</a> Nature. 428, 617-624 (2004).</li>
<li>Goodwin, S., McPherson, J. D., McCombie, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coming+of+age%3A+ten+years+of+next-generation+sequencing+technologies%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">Coming of age: ten years of next-generation sequencing technologies.</a> Nat Rev Genet. 17, 333-351 (2016).</li>
</ol>

作者没有什么可透露的。

虽然这项技术是稳健和相对直接的, 但应特别注意以下方面:
(1) 我们建议, 在达到对数阶段之前, 培养至少12小时, 因为在接种后达到所需密度仅为4小时时, 细胞周期分布的差异明显。我们的假设是, 一个已经达到相对稳定的平衡的细胞周期分布比分布在一个饱和的文化最近转移到新鲜的媒介, 更好地代表一个 "真正的" 日志阶段分布。
(2) 通过将100% 乙醇直接?...

真核基因组复制是在多个离散站点上启动的, 称为复制的起源, 复制分叉在两个方向上进行 (在 Fragkos et、20151中进行了复查)。起源在他们的时间和射击的效率不同, 并且开发了几个技术监测复制起源活动并且阐明这变化的起因。个体起源的活动可以从单链 DNA2的水平推断出来, 它形成在活跃起源附近, 或者通过使用2D 凝胶电泳监测特定的复制中间体3, 两者都可以检测到放射性探针。这两种技术在酵母中比在哺乳动物细胞中更容易应用, 因为起源仅限于前者的特定已知序列。随着微阵列的出现, 可以评估全球发射的起源。这首先是通过标记 DNA 与重同位素, 将细胞从 G1 块释放到含有轻同位素的介质中, 然后在基因组4中监测重光杂交 dna 的形成。高通量测序的引入允许类似的全基因组监测源活动, 而不需要昂贵的同位素标记。根据 dna 含量及其 dna 进行深度测序, 对细胞进行排列。由于序列覆盖率从1x 到2x 在 s 阶段过程中进行, 因此可以通过比较 s 阶段中单元格的读深度与 G1 或 G25、6中的内容来评估相对复制计时。这些技术, 特别是适用于酵母, 导致了更深的理解如何 dna 序列, 染色质结构, 和 dna 复制蛋白调节起源时间和效率。
在细胞增殖过程中, 基因信息的忠实传递不仅需要成功地启动 DNA 复制, 这在起源上发生, 而且复制的成功完成是复制叉相遇的地方。与开始复制一样, 复制完成的时间在整个基因组中都不同, 某些区域甚至在细胞周期的晚期仍未复制。这些区域可能特别远离主动复制的起源, 也可能含有阻碍 DNA 聚合酶的序列或染色质结构。晚期复制区域可以表现为脆弱的站点, 这与染色体断裂和更高的突变率有关, 并且已经牵连到癌症和老化的7,8,9。然而, 尽管正确完成 DNA 复制在维持基因组稳定性方面的重要性, 但我们对其发生地点和方式的理解远远落后于复制启动。虽然研究了晚期复制与疾病有关的个别基因, 例如, qPCR10, 但目前还缺乏旨在阐明晚期复制的地点和根本原因的全球研究。在这里, 我们描述了一种技术, 我们称之为 "G2 序列", 我们结合流式细胞术与高通量测序, 以揭示完成的基因组复制在酵母11。随着小的变化, 这个协议可以适应任何细胞, 可以根据 DNA 的内容流排序。

<table><tbody><tr><td>YeaStar Genomic DNA kit</td><td>Genesee Scientific</td><td>11-323</td><td></td></tr><tr><td>1 &amp;micro;M SYTOX Green</td><td>ThermoFisher</td><td>57020</td><td>resuspend in 50 mM sodium citrate, pH 7.2</td></tr><tr><td>50 mM sodium citrate, pH 7.2</td><td></td><td></td><td></td></tr><tr><td>RNase solution (0.25 mg / mL)</td><td>Sigma</td><td>R6513</td><td>0.25 mg / mL RNaseA resuspended in 50 mM sodium citrate, pH 7.2, boil RNase solution for 10 minutes before the first use only, and from then on store at -20&amp;deg;</td></tr><tr><td>proteinase K solution (20 mg / mL)</td><td>ThermoFisher / Invitrogen</td><td>25530-015</td><td>resuspend in 10 mM Tris, pH 7.5, 2 mM CaCl2, 50% glycerol, store at -20&amp;deg;C</td></tr><tr><td>Model 50 Sonic Dismembrator</td><td>Fisher Scientific</td><td>FB50A220</td><td></td></tr><tr><td>BD Biosciences FACSAria II</td><td>BD Biosciences</td><td>644832</td><td></td></tr><tr><td>Zymo-spin III columns</td><td>Zymo Research</td><td>C1005</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>ThermoFisher</td><td>Q32851</td><td></td></tr><tr><td>Qubit 3.0 Fluorometer</td><td>ThermoFisher</td><td>Q33216</td><td></td></tr><tr><td>Covaris Model LE220 Focused-Ultrasonicator</td><td>Covaris</td><td>500219</td><td></td></tr><tr><td>Illumina TruSeq DNA LT Sample Prep Kit</td><td>Illumina</td><td>15026486</td><td></td></tr><tr><td>Illumina HiSeq 2500 instrument</td><td>Illumina</td><td>SY&amp;ndash;401&amp;ndash;2501</td><td></td></tr><tr><td>gsnap alignment software</td><td>open source software / Genentech</td><td></td><td>http://research-pub.gene.com/gmap</td></tr></tbody></table>

g2-seq: a high throughput sequencing-based technique for identifying late replicating regions of the genome

许多技术已经开发, 以跟踪 DNA 复制的进展, 通过 S 阶段的细胞周期。这些技术大多用于阐明基因组复制的地点和时间, 而不是完成。然而, 至关重要的是, 我们了解基因组中最后一个完成复制的区域, 因为这些区域的染色体断裂和突变水平较高, 并与疾病和衰老有关。在这里, 我们描述了我们如何扩展了一个技术, 已经被用来监测复制启动, 而不是确定这些区域的基因组最后完成复制。这种方法是基于流式细胞术和高通量测序相结合的。虽然本报告着重于酵母的应用, 该方法可以与任何细胞, 可以排序在流式仪根据 DNA 的内容。

我们已经使用上面描述的程序来识别在萌芽的酵母中晚期复制的地点。使用已知的晚期复制区域对人工染色体进行测试, 证明该方法准确可靠。我们的结果还表明, 及时完成复制的生物学重要性表明, 7 号染色体上的一个晚期复制区域在我们的 G2-seq 结果的基础上被识别为晚复制, 大约损失了三倍, 比可比较的控制区域11更频繁。
<p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome at JoVE.com

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

我们描述了一种将流式细胞术和高通量测序相结合的技术来识别基因组的晚期复制区域。

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

g2-seq-high-throughput-sequencing-based-technique-for-identifying

Research

JoVE Journal

Genetics

5.8K 次观看.  Fred Hutchinson Cancer Research Center. 我们描述了一种将流式细胞术和高通量测序相结合的技术来识别基因组的晚期复制区域。

视频: G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

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

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 region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

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 ...

科研

遗传学

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 ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

<meta charset="utf8"></head><body>用于分析 DNA 复制和叉停滞事件的 2D 凝胶电泳

Electrophoretic Analysis of Replication Through Structure-Prone DNA Repeats Within the SV40-Based Human Episome

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This protocol describes how to analyze replication fork progression through structure-prone, expandable DNA repeats within the simian virus 40 (SV40)-based episome in human cells. In brief, upon plasmid transfection into human cells, replication intermediates are isolated by the modified Hirt protocol and treated with the DpnI restriction enzyme to remove non-replicated DNA. Intermediates are then digested by appropriate restriction enzymes to place the repeat of interest within the origin-distal half of a 3-5 kb-long DNA fragment. The replication intermediates are separated into two perpendicular dimensions, first by size and then by shape. Following Southern blot hybridization, this approach allows researchers to observe fork stalling at various structure-forming repeats on the descending half of the replication Y-arc. Furthermore, this positioning of the stall site allows the visualization of various outcomes of repeat-mediated fork stalling, such as fork reversal, the advent of a converging fork, and recombinational fork restart.

<meta charset="utf8"></head><body>作者聚焦:表征致病性重复序列的 DNA 复制以揭示复制叉停滞和扩增的机制

Here, we outline the procedure for analyzing replication progression through pathogenic, structure-prone repeats using 2-dimensional gel electrophoresis.

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

Two-dimensional neutral/neutral gel-electrophoresis (2DGE) emerged as a benchmark technique to analyze DNA replication through natural impediments. This ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

生物工程

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

结合荧光染色体复制时序<em在原位</em&gt;杂交

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

生物学

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

摘要

探索更多视频

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

全基因组对真核生物转录误差的监测

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

从活细胞中的单端粒表达的癌症细胞克隆的生成, 以可视化含有端粒重复的 rna terra

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

通过基于 SV40 的人游离体内易结构 DNA 重复序列的电泳分析进行复制

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

结合荧光染色体复制时序

基因组DNA富集转座为基础的技术的整个序列的农工商分析