Name: atac-seq assay with low mitochondrial dna contamination from primary human cd4+ t lymphocytes
Uploaded: 2019-03-22T14:00:00
Description: Watch this Scientific Journal Video about ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes at JoVE.com

我们感谢 Atsede Siba 的技术支持。c. s. c. 得到国家卫生研究院拨款1R6DA047032 的支持。

这一改进的协议为执行 CD4 + 淋巴细胞的 Ata-seq 提供了分步指导, 从全血的起始材料到数据分析 (图 1)。
1. 全血 CD4+ T 细胞的分离
请注意:该方案的起始材料为15毫升的新鲜全血, 使用标准程序收集, 允许根据研究要求选择起始材料的来源。根据需要扩展协议。预热磷酸盐缓冲盐水 (PBS) + 2% 胎儿小牛血清 (FCS) 至室温 (RT), 并在?...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNase-seq:+a+high-resolution+technique+for+mapping+active+gene+regulatory+elements+across+the+genome+from+mammalian+cells.">DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells.</a> Cold Spring Harbor Protocols. (2), (2010).</li><li>Pajoro, A., Mui&#241;o, J. M., Angenent, G. C., Kaufmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+Nucleosome+Occupancy+by+MNase-seq:+Experimental+Protocol+and+Computational+Analysis.">Profiling Nucleosome Occupancy by MNase-seq: Experimental Protocol and Computational Analysis.</a> Methods in Molecular Biology. 1675, 167-181 (2018).</li><li>Giresi, P. G., Kim, J., McDaniell, R. M., Iyer, V. R., Lieb, J. 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本文介绍的改进的 ATAC-seq 协议提供了可重复的结果, 线粒体 DNA 污染最小。该方案已被用来成功地表征人类原代 Pbmc10、人单核细胞、小鼠树突状细胞 (未发表) 和培养的黑色素瘤细胞系 11的染色质结构。我们预计, 这种改善的裂解条件也有可能适用于其他细胞类型。预计该核分离协议将与单个核 Ata-seq 协议兼容, 最大限度地减少线粒体 DNA 污染, 以改进测序结果。<...

经骨可接触染色质测序 (ATAC-seq) 的检测已迅速成为询问染色质结构的主要方法。ATAC-seq 可以通过标记过程 (用相同的酶对 DNA 进行碎片化和标记) 来识别可访问染色质的区域, 从而生成可测序可以确定整个基因组中可获得染色质的文库。这个标记过程是介导的多活性 Tn5 转座酶, 这只削减开放区域的染色质由于核细胞组位位阻滞。在切割时, tn5 转座酶还插入测序适配器, 通过 pcr 和下一代全基因组可访问染色质 1,2 的测序构建。
ATAC-seq 已成为确定染色质可访问性区域的首选方法, 因为协议相对简单、快速, 质量和信息范围可根据其结果确定, 而且所需的起始材料数量较少。与 DNase-seq3 (也测量全基因组染色质的可及性) 相比, DNase-seq4 (它确定了开放基因组区域中的核糖体位置) 和甲醛介导的 faire-seq5, atac-seq 更快,更便宜, 更可重现性1。它也比较敏感, 与仅有500个原子核的起始材料一起工作, 而 DNase-seq3所需的核为 5, 000万核。与其他方法相比, Atc-seq 还能够提供更多有关染色质结构的信息, 包括转录因子结合、核糖体定位和开放染色质区域1。有效的单细胞 atc-seq 协议已经得到验证, 提供了关于单细胞级别6,7的染色质结构的信息。
Ata-seq 已被用于描述染色质结构的范围广泛的研究和细胞类型, 包括植物8, 人类9, 和许多其他生物。它在确定疾病状态的表观遗传调控方面也至关重要。然而, 最广泛使用的 Ata-seq 协议包括从线粒体 DNA 读取污染测序的主要缺点。在某些数据集中, 此污染水平可能高达测序结果1的60%。在这一领域作出了协调一致的努力, 以减少这些污染的线粒体读数, 从而能够更有效地应用 ata-seq 7、10、11.在这里, 我们提出了一个改进的 Ata-seq 协议, 将线粒体 DNA 污染率降低到只有 3%, 允许减少约50% 的测序成本 10。这是通过简化的 CD4 + 淋巴细胞分离和活化过程以及改进的裂解缓冲液实现的, 这对于最大限度地减少线粒体 DNA 污染至关重要。
这种增强型 Ata-seq 协议已通过广泛的原代细胞得到验证, 包括人类原代外周血单个核细胞 (Pbmc) 10、人原代单核细胞和小鼠树突状细胞 (未发表)。它还被成功地用于在非编码元素11的聚集定期间隔短回文重复 (CRISPR) 屏幕中询问黑色素瘤细胞系。此外, 本协议中描述并在 GitHub 上提供的数据分析包为新的、有经验的研究人员提供了分析 Ata-seq 数据的工具。ATAC-seq 是在整个基因组中绘制染色质可访问性的最有效的检测方法, 对此处介绍的现有协议进行修改将使研究人员能够生成具有低线粒体 DNA 污染的高质量数据,降低测序成本, 提高 Ata-seq 吞吐量。

<table>
	<tbody>
		<tr>
			<td>1X PBS, Sterile</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>5810/5810&#160;R Swing Bucket Refrigerated Centrifuge with 50 mL, 15 mL, and 1.5 mL Tube Buckets</td>
			<td>Eppendorf</td>
			<td>22625501</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>96 Well Round Bottom Plate</td>
			<td>Thermo Scientific</td>
			<td>163320</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Agilent 4200 Tape Station System</td>
			<td>Agilent</td>
			<td>G2991AA</td>
			<td>Suggested for quality assessment.</td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>374081</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Dynabeads Human T-Activator CD3/CD28</td>
			<td>Invitrogen Life Technologies</td>
			<td>11131D</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Dynabeads Untouched Human CD4 T Cells Kit</td>
			<td>Invitrogen Life Technologies</td>
			<td>11346D</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Dynamagnet</td>
			<td>Invitrogen Life Technologies</td>
			<td>12321D</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Gemini Bio Products</td>
			<td>100-500</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>High Sensitivity DNA Kit</td>
			<td>Agilent</td>
			<td>50674626</td>
			<td>Suggested for quality assessment.</td>
		</tr>
		<tr>
			<td>Magnesium Chloride, Hexahydrate, Molecular Biology Grade</td>
			<td>Sigma</td>
			<td>M2393</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>MinElute PCR Purification Kit (Buffer PB, Buffer PE, Elution Buffer)</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Thermo Scientific</td>
			<td>5100-0001</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>NaCl, Molecular Biology Grade</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>NEBNext High Fidelity 2X PCR Master Mix</td>
			<td>New England BioLabs</td>
			<td>M0541</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Nextera DNA Library Preparation Kit (2X TD Buffer, Tn5 Enzyme)</td>
			<td>Illumina</td>
			<td>FC1211030</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Nuclease Free Sterile dH20</td>
			<td>Gibco</td>
			<td>10977015</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween20)</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>PowerUp SYBR Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td>A25780</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Precision Water Bath</td>
			<td>Thermo Scientific</td>
			<td>TSGP02</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Invitrogen Life Technologies</td>
			<td>Q32851</td>
			<td>Suggested for quality assessment.</td>
		</tr>
		<tr>
			<td>Qubit FlouroMeter</td>
			<td>Invitrogen Life Technologies</td>
			<td>Q33226</td>
			<td>Suggested for quality assessment.</td>
		</tr>
		<tr>
			<td>Rosette Sep Human CD4+ Density Medium</td>
			<td>Stem Cell Technologies</td>
			<td>15705</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Rosette Sep Human CD4+ Enrichment Cocktail</td>
			<td>Stem Cell Technologies</td>
			<td>15022</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Sterile Resevoir</td>
			<td>Thermo Scientific</td>
			<td>8096-11</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>T100 Thermocycler with Heated Lid</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Tris-HCL</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>Can use other comparable products.</td>
		</tr>
	</tbody>
</table>

atac-seq assay with low mitochondrial dna contamination from primary human cd4+ t lymphocytes

ATAC-seq 由于其快速简便的方法绘制了全基因组可访问的染色质, 已成为表观遗传学研究中广泛使用的一种方法。在本文中, 我们提出了一个改进的 Ata-seq 协议, 减少污染线粒体 DNA 读取。虽然以前的 Ata-seq 协议与平均50% 的污染线粒体 DNA 的操作作斗争, 但该协议中引入的优化裂解缓冲液将线粒体 DNA 污染降低到平均3%。这种改进的 Ata-seq 协议使测序成本降低了近50%。我们展示了如何从激活的 CD4 + 淋巴细胞制备这些高质量的 Ata-seq 库, 通过数据分析提供 CD4 + 淋巴细胞从全血分离的分步指导。这种改进的 Atc-seq 协议已在广泛的细胞类型中得到验证, 并将立即用于研究染色质可获得性的研究人员。

从15毫升的新鲜全血, 该协议产生平均 100万个 CD4 + T 细胞。这些可以冻结以后处理或立即使用。CD4+ T 细胞的活力, 新鲜或解冻, 持续 & gt;95。这种 CD4+ T 细胞分离方法允许在源材料和收集时间方面具有灵活性。这种改进的 Ata-seq 协议生成了一个大于 1 ngμl 的最终库, 用于测序。使用市售系统进行的质量控制应显示200至 1, 000 bp 之间的 DNA 片段 (图 2)。只能...

Watch this Scientific Journal Video about ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes at JoVE.com

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

在这里, 我们提出了一个协议, 以执行检测转座受接触染色质测序 (ATAC-seq) 激活 CD4 + 人类淋巴细胞。该协议已被修改, 以尽量减少污染线粒体 DNA 读取从50% 到3% 通过引入一个新的裂解缓冲液。

原发性人类 CD4+ T 淋巴细胞低线粒体 DNA 污染的 ATAC-seq 检测

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

这种经过改进的 ATAC-seq 协议引入了一种新的解压缓冲液，可将线粒体读取从 50% 降至 3% 以下。线粒体污染的减少将使研究人员能够执行更高效的 ATAC-seq，测序成本降低 50%。我们已经在人类原发性PBMC、人类原生单核细胞、小鼠树突状细胞、黑色素瘤细胞系中验证了这种新的AC-seq协议。我们相信它将在广泛的细胞类型中有效。在细胞激活和选择过程中最大限度地减少样品损失对于使用 ATAC-seq 获得高质量结果至关重要。我们将演示如何使用基本实验室设备处理来自珍贵样品的少量细胞。首先，在37摄氏度的水浴中解冻一小瓶以前分离的CD4+T细胞，直到冰刚刚融化。然后轻轻地将细胞转移到一个15毫升的圆锥形管中，该管含有9毫升预加热的完整RPMI。将细胞在1500倍的G和4摄氏度的离心，6分钟。然后丢弃上一提液，轻轻地将颗粒重新在两毫升完整的 RPMI 中。重复离心后，丢弃上经剂，并轻轻地在0.5毫升的完整RPMI中重新暂停细胞。使用血细胞计计算细胞数。使用完整的 RPMI，将电池悬浮液密度调整为板 50，000 CD4+细胞，每井 96 井圆底板的 200 微升。然后，准备磁珠，等价12.5微升的人类T-Activator CD3/CD28珠子每个AC-seq样品到1.5毫升管。将1毫升1x PBS加入管子，放在磁铁上一分钟清洗珠子。小心地拆下并丢弃透明上清剂，然后从磁铁上拆下管子。每个 ATAC-seq 样品将珠子重新在 13 微升中，提供完整的 RPMI。要使用调整后的浓度激活 CD4+T 细胞，请将 500，000 个细胞收集在 15 毫升圆锥形管中 2.1 毫升的完整 RPMI 介质中。加入12.5微升的人类T-Activator珠子，在前几步准备和清洗，然后通过反转管子轻轻混合。然后用珠子轻轻地将细胞转移到无菌储液罐中。使用多通道移液器，板200微升细胞与珠子每井的圆底96井板，并在37摄氏度，5%CO2培养箱48小时孵育。完成孵育后，在423倍G和室温下将板离心8分钟。从每孔中去除100微升的介质，在丢弃前将其收集在锥形管中，以确认没有珠子损失。然后将细胞颗粒重新在剩余的100微升的介质中，并在1.5毫升的管中收集所有内容。要分离CD4+细胞，在一毫升完整的RPMI中，将50微升预洗的CD4结合珠子加入50万个细胞中，然后反转混合。在4摄氏度下孵育20分钟，同时每6分钟用手轻拂一次混合。将管子放在磁铁上两分钟，直到上清液清除，然后取出并丢弃。从磁铁上拆下管子。将珠状细胞重新用一毫升PBS和2%FCS洗净，将管子放回磁铁上一分钟。丢弃透明上清剂，并重复此过程，总共三次洗涤。最后洗涤后，将管子放在磁铁上一分钟。取出它，丢弃上一液，然后将颗粒放在冰上。接下来，进行核分离，将珠子和细胞重新在50微升的冷解解缓冲液中，并立即在500G、4摄氏度的离心机中，10分钟。取出并丢弃上一液。要执行转置酶反应，用50微升Tn5转置酶混合物重新暂停分离的核颗粒。在37摄氏度下在热循环器中孵育30分钟，盖子设定为40摄氏度，完成后在4摄氏度下保持。热循环后，立即执行快速台式离心机旋转。将管子放在磁铁上一分钟，从产品中去除珠子。在不从磁铁上去除管，将透明上清液转移到DNA纯化柱。用 250 微升 PB 缓冲器清洗一次柱，用 750 微升 PE 缓冲器洗两次。然后添加 10 微升洗脱缓冲液来洗脱样品。在无核酸酶PCR管中设置初始PCR扩增反应。将 PCR 管放入热循环器中，然后运行 PCR 扩增程序。然后完成剩余 45 微升 PCR 反应的 PCR 扩增，如手稿中所述。将 PCR 管放在热循环器中并运行程序。完成放大后，使用 PCR 清理套件按照制造商的协议进行净化库，在洗脱缓冲液的 25 微升中洗脱。将下一代测序器上准备好的库按每个样本的平均读取深度 4200 万次。这种改进的 ATAC-seq 协议产生了一个最终库，每个微升的纳米图大于一个，用于测序。质量控制显示DNA片段在200到1000个碱基对之间，并且通过这些高质量的库进行了进一步的测序。按照该协议编写的库显示只有3%的线粒体污染。在生物复制中，可用读取的高百分比是足够的恒定的。该协议在技术复制和生物复制之间提供了高度可重复的结果。此外，该协议需要 48 小时，而不是前面描述的一周或一周以上，从而实现一致和高效的激活，如可重现的测序结果所证明的那样。此外，分析管道准确地调用了预测的 ATAC-seq 峰值。测序结果分析显示，在人类T细胞活化期间，染色质状态发生了明显变化。在激活前和激活后48小时内，在6个样品之间确定了开放染色质的可分色可访问区域。确保用冷冻试剂和材料进行核解解反应，以最大限度地回收完整的核。我们改良的核解液缓冲液对细胞更温和，并最大限度地减少了线粒体DNA的污染。我们期待这种经过修改的解液缓冲液也应用于单核 ATAC-seq。我们知道，该协议将允许研究人员以降低成本的价格生成更高质量的 ATAC-seq 数据，从而帮助推进表观遗传领域。该协议简单、快速，不需要任何危险试剂或仪器，因此很容易为 ATAC-seq 的新功能获取。

Research

JoVE Journal

Genetics

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

科研

遗传学

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 ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

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

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 ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

伦蒂病毒介导基因在低附着板中制备的人类伪性沉默

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

从小鼠横纹肌瘤分离的原发性肿瘤细胞的肿瘤球衍生和治疗

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

微卫星DNA基因分型和流细胞学比较分析形式固定石蜡嵌入水合体摩尔组织

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are ...

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

3D多色DNAFISH工具研究人类原细胞核结构

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...