Name: g2-seq: a high throughput sequencing-based technique for identifying late replicating regions of the genome
Uploaded: 2018-03-22T12:30:00
Description: 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

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))?...

<ol>
	<li>Fragkos, M., Ganier, O., Coulombe, P., Mechali, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+origin+activation+in+space+and+time.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mec1-+and+Rad53-dependent+checkpoint+controls+late-firing+origins+of+DNA+replication.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+replication+fork+barrier+at+the+3'+end+of+yeast+ribosomal+RNA+genes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+dynamics+of+the+yeast+genome.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+replication+timing+reveals+global+and+local+regulation+of+replication+origin+activity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRC1-dependent+scaling+of+the+budding+yeast+DNA+replication+timing+program.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+rates+across+budding+yeast+chromosome+VI+are+correlated+with+replication+timing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+fragile+sites.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+instability+at+chromosomal+fragile+sites+in+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very+late+DNA+replication+in+the+human+cell+cycle.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIR2+suppresses+replication+gaps+and+genome+instability+by+balancing+replication+between+repetitive+and+unique+sequences.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GMAP+and+GSNAP+for+Genomic+Sequence+Alignment:+Enhancements+to+Speed,+Accuracy,+and+Functionality.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+a+flexible+suite+of+utilities+for+comparing+genomic+features.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aligning+short+sequencing+reads+with+Bowtie.">Aligning short sequencing reads with Bowtie.</a> Curr Protoc Bioinformatics. , 17 (2010).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proof+and+evolutionary+analysis+of+ancient+genome+duplication+in+the+yeast+Saccharomyces+cerevisiae.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coming+of+age:+ten+years+of+next-generation+sequencing+technologies.">Coming of age: ten years of next-generation sequencing technologies.</a> Nat Rev Genet. 17, 333-351 (2016).</li></ol>

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 width="722">
	<tbody>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">YeaStar Genomic DNA kit</td>
			<td style="width:135px;">Genesee Scientific</td>
			<td style="width:119px;">11-323</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">1 &#181;M SYTOX Green</td>
			<td style="width:135px;">ThermoFisher</td>
			<td style="width:119px;">57020</td>
			<td>resuspend in 50 mM sodium citrate, pH 7.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">50 mM sodium citrate, pH 7.2</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="width:301px;height:147px;">RNase solution (0.25 mg / mL)</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">R6513</td>
			<td style="width:167px;">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&#176;</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:301px;height:42px;">proteinase K solution (20 mg / mL)</td>
			<td style="width:135px;">ThermoFisher / Invitrogen</td>
			<td style="width:119px;">25530-015</td>
			<td>resuspend in 10 mM Tris, pH 7.5, 2 mM CaCl2, 50% glycerol, store at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Model 50 Sonic Dismembrator</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>FB50A220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">BD Biosciences FACSAria II</td>
			<td style="width:135px;">BD Biosciences</td>
			<td>644832</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Zymo-spin III columns</td>
			<td style="width:135px;">Zymo Research</td>
			<td style="width:119px;">C1005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:135px;">ThermoFisher</td>
			<td style="width:119px;">Q32851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Qubit 3.0 Fluorometer</td>
			<td style="width:135px;">ThermoFisher</td>
			<td style="width:119px;">Q33216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Covaris Model LE220 Focused-Ultrasonicator</td>
			<td style="width:135px;">Covaris</td>
			<td style="width:119px;">500219</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Illumina TruSeq DNA LT Sample Prep Kit</td>
			<td style="width:135px;">Illumina</td>
			<td>15026486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:301px;height:21px;">Illumina HiSeq 2500 instrument</td>
			<td style="width:135px;">Illumina</td>
			<td>SY&#8211;401&#8211;2501&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:301px;height:63px;">gsnap alignment software</td>
			<td style="width:135px;">open source software / Genentech</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">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。

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