Name: in-nucleus hi-c in drosophila cells
Uploaded: 2021-09-15T15:00:00
Description: Watch this Scientific Journal Video about In-Nucleus Hi-C in Drosophila Cells at JoVE.com

这项工作得到了联阿特派团技术创新和研究支助方案（PAPIIT）赠款编号为207319和科学技术国家委员会（CONACyT-FORDECyT）赠款编号303068的支持。A.E.-L.是国家科学技术委员会（CONACyT）CVU号码968128支持的硕士生。

1. 固定
<ol><li>从 1000 万施耐德的 2 号线加 （S2R+） 细胞开始，在施耐德介质中准备 17.5 mL 的细胞悬浮，其中含有 10% 的胎儿牛血清 （FBS） 在室温下 （RT）。</li>
	<li>加入无甲醇甲醛，最终浓度为2%。混合和孵育10分钟在RT，小心混合每分钟。 注：甲醛是一种危险化学品。遵守相应的健康和安全法规，并在烟罩中工作。</li>
	<li>通过添加甘氨酸来抑制反应，实现最终浓度为 0.125 M 和混合。在 Rt 孵?...

<ol>
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这里介绍的核内Hi-C方法允许以高分辨率详细探索果蝇基因组拓扑学，提供不同基因组尺度的基因组相互作用的视图，从促进剂和增强剂等调控元素之间的染色质循环到TAD和大隔间识别25。同样的技术也有效地应用于哺乳动物组织与一些修改33。例如，当处理组织而不是单细胞悬浮时，组织通过 70 μm 过滤器进行筛选，裂解步骤在使用 Dounce 同质化器对材料进行均?...

在真核生物中，基因组被分割成染色体，在相间第1阶段占据核空间的特定区域。形成染色体的染色质可分为两种主要状态：一种是转录允许的可访问染色质，另一种是转录抑制的紧凑型染色质。这些染色质状态在核空间中分离和很少混合，在核2中形成两个不同的隔间。在亚兆基尺度上，边界分隔高频染色质相互作用的领域，称为TAD，标记染色体组织3，4，5。在哺乳动物中，TAD边界被凝聚力和CCTC结合因子（CTCF）6、7、8所占据。凝固素复合物挤出色度素，并停止在CTCF结合点，在基因组序列的收敛方向处置，形成稳定的染色质循环9，10，13，14。CTCF DNA结合部位点在CTCF和凝聚蛋白丰度的边界上发生基因破坏或减少，导致调控元素之间的异常相互作用、TAD形成的丧失和基因表达放松管制9、10、11、13、14。
在德罗索菲拉， TAD 之间的边界由多个 AP 占据，包括边界元素相关因子 32 kDa （BEAF-32）、Motif 1 结合蛋白 （M1BP）、中生代蛋白 190 （CP190）、毛翼抑制剂 （SuHW） 和 CTCF，并富含活性组蛋白修饰和聚合酶 II 16、17、18。有人建议，在德罗索菲拉，TADs出现的结果是转录13，17，19，和独立的AP在边界形成和绝缘属性的确切作用仍在调查之中。因此，德罗索菲拉的域名是否是类似转录状态区域聚合的唯一结果，或者包括反恐委员会在内的 AP 是否有助于边界形成，仍有待充分说明。通过开发染色体构象捕获技术以及下一代测序，可以探索高分辨率基因组接触。Hi-C 协议首先描述为"解决方案"2的粘合步骤，以避免染色质片段之间的虚假粘合产品。然而，一些研究指出，数据中的有用信号来自在部分赖化核形成的结结产品，这些结核不在解决方案20、21中。
然后，作为单细胞Hi-C实验22的一部分，对协议进行了修改，以在细胞核内进行粘合。核内Hi-C协议随后被纳入细胞群Hi-C，在全基因组距离内产生更一致的覆盖，并产生技术噪声较少的数据23，24。该协议，在这里详细描述，是基于人口在细胞核Hi-C协议23，24，并用于调查遗传去除DNA结合图案的CTCF和M1BP的后果，从领域边界在D罗索菲拉25。结果表明，改变边界上对 APs 的 DNA 结合图案对 Notch 域的形成、Notch点周围区域更大的拓扑缺陷以及基因表达放松管制有严重的影响。这表明，域界的遗传元素对于维持25号果蝇的基因组拓扑学和基因表达非常重要。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>16% (vol/vol) paraformaldehyde solution</td>
			<td>Agar Scientific</td>
			<td>R1026</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-14-dATP</td>
			<td>Invitrogen</td>
			<td>CA1524-016</td>
			<td></td>
		</tr>
		<tr>
			<td>ClaI enzyme</td>
			<td>NEB</td>
			<td>R0197S</td>
			<td></td>
		</tr>
		<tr>
			<td>COVARIS Ultrasonicator</td>
			<td>Covaris</td>
			<td>LE220-M220</td>
			<td></td>
		</tr>
		<tr>
			<td>Cut Smart</td>
			<td>NEB</td>
			<td>B72002S</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) 1x</td>
			<td>Life Technologies</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptabidin C1</td>
			<td>Invitrogen</td>
			<td>65002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS) sterile filtered</td>
			<td>Sigma</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow Dna PolI large fragment</td>
			<td>NEB</td>
			<td>M0210L</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow exo(-)</td>
			<td>NEB</td>
			<td>M0210S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ligation Buffer</td>
			<td>NEB</td>
			<td>B020S</td>
			<td></td>
		</tr>
		<tr>
			<td>MboI enzyme</td>
			<td>NEB</td>
			<td>R0147M</td>
			<td></td>
		</tr>
		<tr>
			<td>NP40-Igepal</td>
			<td>SIGMA</td>
			<td>CA-420</td>
			<td>Non-ionic surfactant for addition in lysis buffer</td>
		</tr>
		<tr>
			<td>PE adapter 1.0</td>
			<td>Illumina</td>
			<td>5&#39;-P-GATCGGAAGAGCGGTTCAGCAG 
			GAATGCCGAG-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE adapter 2.0</td>
			<td>Illumina</td>
			<td>5&#39;-ACACTCTTTCCCTACACGACGCT 
			CTTCCGATCT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE PCR primer 1.0</td>
			<td>Illumina</td>
			<td>5&#39;-AATGATACGGCGACCACCGAGAT 
			CTACACTCTTTCCCTACACGACG 
			CTCTTCCGATCT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>PE PCR primer 2.0</td>
			<td>Illumina</td>
			<td>5&#39;-CAAGCAGAAGACGGCATACGAG 
			ATCGGTCTCGGCATTCCTGCTGA 
			ACCGCTCTTCCGATCT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol: Chloroform:Isoamyl Alcohol 25:24:1</td>
			<td>SIGMA</td>
			<td>P2069</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 1 (known interaction, Figure 2A)</td>
			<td>Sigma</td>
			<td>5&#39;-TCGCGGTAATTTTGCGTTTGA-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer 2 (known interactions, Figure 2A)</td>
			<td>Sigma</td>
			<td>5&#39;-CCTCCCTGCCAAAACGTTTT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablet</td>
			<td>Roche</td>
			<td>4693132001</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche</td>
			<td>3115879001</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit</td>
			<td>ThermoFisher</td>
			<td>Q33327</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse</td>
			<td>Roche</td>
			<td>10109142001</td>
			<td></td>
		</tr>
		<tr>
			<td>SPRI Beads</td>
			<td>Beckman</td>
			<td>B23318</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Invitrogen</td>
			<td>15224-025</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA polymerase</td>
			<td>NEB</td>
			<td>M0203S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase (PNK)&#160;</td>
			<td>NEB</td>
			<td>M0201L</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqPhusion</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td>DNA polymerase</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>Non-ionic surfactant for quenching of SDS</td>
		</tr>
	</tbody>
</table>

in-nucleus hi-c in drosophila cells

基因组被组织成拓扑关联域（TADs），由隔离域之间相互作用的边界划分。在德罗索菲拉，TAD形成和边界背后的机制仍在调查中。此处描述的核内 Hi-C 方法的应用有助于解剖 TAD 边界下隔离 诺奇 基因的建筑蛋白 （AP） 结合位点的功能。导致 APs 损失的域界的基因改造会导致 TAD 融合、转录缺陷和远程拓扑变化。这些结果提供了证据，证明遗传元素对果蝇的域界形成和基因表达控制的贡献。在这里，详细描述了核内Hi-C方法，它提供了重要的检查点，以评估实验的质量以及协议。还显示所需的测序读数和有效的 Hi-C 对，以分析不同基因组尺度的基因组相互作用。CRISPR/Cas9介质的遗传编辑调节元素和高分辨率的基因组相互作用分析使用这种核内Hi-C协议可以是一个强大的组合，研究遗传元素的结构功能。

下面描述的是一个成功的高C协议的结果（见图1A中的高C协议工作流程摘要）。在核中Hi-C实验期间，有几个质量控制检查点。样品在 （UD） 和 （D） 染色素限制步骤之前和之后以及连合 （L） 之后收集。十字线被逆转，DNA被净化，运行在阿加罗斯凝胶上。当与Mbo I的限制成功时，观察到200-1000个基点的涂片（图1B）。分子的预期大小取决于选择的限制酶?...

Watch this Scientific Journal Video about In-Nucleus Hi-C in Drosophila Cells at JoVE.com

In-Nucleus Hi-C in Drosophila Cells

基因组在核空间中组织成不同的结构，可以通过染色体构象捕获技术揭示出来。核内Hi-C方法提供了在果蝇细胞系中全基因组的染色质相互作用集合，生成接触图，可以在限制片段水平下以兆基分辨率进行探索。

果蝇细胞中的核内高C

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, ...

核Hi-C协议允许以不同分辨率探索染色体确认，以描述组织基因组的染色质环、域和隔间。此协议提供不同尺度的基因组相互作用的高分辨率分析，在全范围基因组距离和具有较少技术噪声的数据上实现更一致的覆盖。将此协议与基因编辑相结合是测试遗传元素结构功能的有力策略。它也可以应用于导致疾病的结构变异的表征。核Hi-C协议可用于探索任何真核基因组的基因组组织。做SDS和Triton处理通过管道分解任何核团块，因为聚合物干扰酶反应效率，并确保在测序前执行适当的质量控制措施。首先，在施耐德介质的17.5毫升中加入1倍于10倍的无甲醇甲醛，在17.5毫升施耐德介质中加入果蝇细胞，最后浓度为2%，在室温下将细胞孵化10分钟，每60秒混合一次，或在旋转轮上混合一次， 并将甘油加入到混合的 0.125 摩尔的最终浓度中。在室温下5分钟和冰上15分钟后，通过离心收集细胞，并小心地将颗粒重新浸入25毫升的冷PBS中，并用另一个离心器收集细胞。离心结束时，将细胞重新吸纳在一毫升冰冷裂解缓冲器中，用于计数，并将细胞调整为每毫升浓度的1倍至6个细胞。将细胞在冰上孵育30分钟，每两分钟倒置一次管子，用另一个离心器混合和收集细胞。将颗粒重新悬浮在一毫升新鲜冰冷裂解缓冲器中。离心后，用一毫升1.25倍的限制缓冲器清洗裂解液。再次离心收集解液。将颗粒重新填充在 360 微升的新鲜限制缓冲器和 11 微升的 10% SDS 中，小心地在 37 摄氏度下管道和孵育 45 分钟，每分钟 7 到 950 转，偶尔吹笛。在孵化结束时，停止用 75 微升非离子表面活性剂的渗透，并将管子返回孵化器再 45 分钟，偶尔吹笛以每分钟 950 转的速度摇晃。对于色素消化，在37摄氏度的旋转下，在管中加入200单位Mbo1，进行4至16小时的孵化。在孵化结束时，在60摄氏度下用20分钟的潜伏灭活酶。要填充限制片段悬垂和标记DNA结束与生物素， 加入1.5微升每10毫摩尔dCTP，dGTP，dTTP，20微升0.4毫摩尔生物素dATP，17.5微升三低EDTA缓冲器，和10微升5单位每微升DNA聚合酶一个大片段到管仔细混合。在37摄氏度下将反应孵化75分钟，每10秒以每分钟700转的速度颤抖30秒。在孵化结束时，将结扎混合物添加到染色质中，并将其调整到一毫升最终反应体积，在 16 摄氏度下进行彻底的温和混合和孵育过夜。为了降解样品蛋白质，在37摄氏度的潜伏中加入50微升每毫升10毫克的蛋白质酶K。在孵化结束时，将温度在夜间升高至65摄氏度，以扭转样品的交联。第二天早上，在37摄氏度下一小时后，用每毫升10毫克的10微升降解RNA，在管中加入一个体积的酚氯仿，通过反转彻底混合，获得同质白相。根据标准协议沉淀DNA后，使用有选择地与DNA结合的荧光染料和根据制造商说明的荧光仪对DNA进行量化。为了从未消化和消化的碱液中清除DNA，将100毫微克未消化、消化和贴合的样品加载到1.5%的玫瑰凝胶上，并在消化的样品中寻找以500个碱基对为中心的涂片，而在消化的样品中寻找一个高分子量带。要通过放大和消化已知的相互作用来验证 Hi-C 标记和粘合效率，PCR 之后，用 Mbo1、Cla1 或两者兼有地消化产品，并在新的 1.5 到 2% 凝胶上运行样品，以便估计 3C 和高 C 结的相对数量。对样本进行测定后，获得200至500个碱基对DNA片段， 将每个样品中含有5微克DNA的130微升微升导管转移到新的微中心管中，其中含有16个10倍结结缓冲器的微升，2个10毫摩尔DATP微升，5个T4DNA聚合酶微升，以及7个双蒸馏水微升管，在20摄氏度下孵育30分钟。在孵化结束时，在管子中加入5个10毫摩尔dNTP的微升、4个10倍结结缓冲的微升、5个T4多核苷酸激酶微升、1个DNA聚合酶的微升1个大片段和25个双蒸馏水微升，在20摄氏度下进行第二次30分钟的孵化。要选择主要在 250 到 550 基对大小范围内的片段，则使用 0.6 倍执行顺序固体相可逆和动员大小选择，然后根据制造商的说明选择 0.9 倍，然后使用 100 微升的三色低 EDTA 提取 DNA。对于每个库的生物素下拉，用 400 微升 1X Tween 缓冲器两次清洗 150 微升与斯特雷普塔维丁相连的磁珠，并在旋转轮上旋转样品三分钟。在孵化结束时，将珠子重新用 300 微升的 2 倍无 Tween 缓冲器中重新使用，并与 300 微升高 C 材料混合，在旋转轮上旋转 30 分钟。在孵化结束时，用 400 微升 0.5 倍 Tween 缓冲器清洗珠子，在 55 摄氏度和每分钟 750 转时进行三分钟的孵化，然后在 200 微升中洗两次，每次洗涤 1 倍限制缓冲。第二次洗涤后，在 100 微升 dATP 尾矿混合物中重新吸收珠子，在 37 摄氏度下孵育 30 分钟。在孵化结束时，将珠子重新注入1X结膜缓冲器的50微升中，并将悬浮液转移到一个新的管子中，该管包含4个微升前配对端适配器和2个T4韧带微升。在室温下两小时后，取出超强的珠子，用400微升的特温缓冲器洗珠两次，然后用200微升无特温缓冲器洗珠子，用100微升限制缓冲器洗珠子，然后用1倍限制缓冲器的40微升重新悬挂珠子。当 Mbo1 限制成功时，会观察到 200 到 1000 个碱基对的涂片。如果连合成功，在凝胶顶部观察到高分子量带。消化效率也可以通过定量PCR得到证实。可接受的消化效率为 80% 或更高。如前所述，通过消化放大中回收的 PCR 产品，可以估算高 C 粘合产品中已知中距离相互作用的放大。建议消化效率超过 70%，以避免在测序后为图书馆提供大量非有用的读物。最终放大的 PCR 周期数应少于可见涂片的周期数。图书馆的消化水平表明有效高C对的丰富性，并反映了从图书馆获得的有用读物的比例。使用唯一有效的高 C 对，可以对配对分布进行基本分析。正如在这个具有代表性的例子中观察到的，NOTCH基因位点与建筑蛋白、域一和二以及沿位点的组蛋白修饰一起可见。删除包含 CTCF 和 M1BP DNA 结合位点的区域后，可以看到染色质接触的戏剧性变化。在 Hi-C 实验之后，可以执行一个可以达到特定的接触集，例如，来自发起人区域。这在评估大型基因组时特别有用。如前所示，将Hi-C协议与基因添加相结合可用于评估基因组元素的结构和调控功能。

Research

JoVE Journal

Genetics

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

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

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

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Quantifying Abdominal Pigmentation in Drosophila melanogaster

量化腹部色素沉着<em&gt;黑腹果蝇</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

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

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 ...

Purification of Low-abundant Cells in the Drosophila Visual System

果蝇视觉系统中低丰细胞的纯化

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

在Vivo功能研究的疾病相关的稀有人类变异使用果蝇

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Use of Drosophila S2 Cells for Live Imaging of Cell Division

使用果蝇S2细胞进行细胞分裂的活成像

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

A FACS-based Protocol to Isolate RNA from the Secondary Cells of Drosophila Male Accessory Glands

从果蝇雄性附件腺的二级细胞中分离RNA的FACS协议

To understand the function of an organ, it is often useful to understand the role of its constituent cell populations. Unfortunately, the rarity of ...

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

使用DNA纤维测定检测人细胞中复制后间隙的积累和修复

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...