a.1 得到加拿大卫生研究院的加拿大研究生奖学金的支持。这项工作得到了来自不列颠哥伦比亚省和加拿大卫生研究院 (CIHR-120589) 的赠款的支持, 这是加拿大的遗传学、环境和健康研究联合会倡议的一部分, 也是由特里福克斯研究所计划提供的。项目赠款 #TFF-122869 M.H. 和一个特里福克斯研究所新的研究员奖 (授予 1039) 到 M.H。

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作者声明他们没有竞争的金融利益。

考虑到染色质修饰和核定位在转录调控中的组合性质, 一种能够同时测量这些特性的方法可能会为表观遗传学的调控提供新的见解。这里介绍的 ndChIP-seq 协议是一种本地芯片-seq 协议, 用于在稀有的主单元中同时对组蛋白修饰和核密度进行审讯,9,10。ndChIP-seq 利用酶消化染色质, 当耦合到配对结束大规模并行排序和高斯混合分布模型, 允许调查组蛋白修饰在单一的核水平和由种群内异质性驱动的表剖面的反褶积。使用此协议, 我们以前报告了免疫沉淀片段大小的唯一分布, 由配对结束读取边界确定, 与 ChromHMM10,24定义的特定染色质状态相关。
ndChIP 序列库的质量取决于多种因素, 如抗体特异性和敏感性、最佳 MNase 消化条件和染色质质量。使用的抗体的特异性是关键的生产成功的 ndChIP-seq 库。一个理想的抗体显示高亲和性的表位的兴趣与小交叉反应性与其他抗原。同样重要的是选择磁性珠与最高亲和力的抗体选择。
MNase 消化是该协议中的一个关键和时间浓度敏感的反应。因此, 在处理多个样本时, 每个反应都必须在相当的时间内孵化 (参见步骤 2.2)。染色质的质量是另一个因素, 显着影响的结果 ndChIP-seq. 分裂染色质导致一个次优 MNase 消化剖面和结果在低信噪比的图书馆。本协议不推荐使用低细胞活性或降解染色质的初级样品, 如福尔马林固定石蜡 (FFPE) 组织。
在染色质提取过程中添加 PIC 可以减少不受欢迎的 (即, 随机) 染色质碎片, 并保留组蛋白尾部的完整性。因此, PIC 需要添加到裂解缓冲区和沉淀缓冲区, 才使用。在通过流式细胞仪选择细胞时, 选择可行的细胞, 并确保细胞以较低的流速排列, 以提高细胞数估计的准确性和生存能力。避免将单元格直接分类到裂解缓冲区。鞘缓冲液会稀释裂解缓冲液, 防止细胞膜的有效性 MNase。根据类型的细胞或生物体, 滴定 MNase 可能需要获得最佳的消化。
哺乳动物细胞的 ndChIP-seq 需要最小测序深度为5000万配对读 (2500万片段) 为狭窄的标记和1亿配对读 (5000万片断) 为宽广的标记和输入。高斯混合分布算法对已被测序到深度显著低于本建议的库, 将无法达到最佳性能。ndChIP-seq 将不分类促进者, 很少分离之间的加权分布值的单一和 di 核片段长度成单或 di 核主导的发起人。因此, 在随后的分析中必须删除这些启动子。可以生成生物复制, 以增加对预测分布的信心, 并确定 MNase 消化和图书馆建设中的技术变异性。
与本机芯片序列协议的以前的迭代不同, ndChIP 提供了通过利用片段大小后沉淀来整合核密度来研究染色质结构和组蛋白修饰的组合效应的方法,由 MNase 的可及性决定, 组蛋白修饰测量。ndChIP 在原发性细胞和组织中的应用, 将为表观遗传调控的综合性质提供新的见解, 并能识别由于群体内异质性而导致的后生特征。

真核细胞基因组被包装成染色质通过重复核结构, 包括两个副本的四组蛋白 (例如, H2A, H2B, H3, 和 H4) 的限制由146基对的 DNA1,2。染色质重塑复合体在基因启动子边界内控制核位置, 并通过改变 DNA 对转录因子的可及性来参与基因表达的调控, 并对 RNA 聚合酶机械3, 4。
核内组蛋白的氨基末端尾端受到各种共价键的修饰, 包括乙酰化、甲基、磷酸、化、sumo、甲, 以及特定氨基酸的羟氨酸5,6,7,8. 这些修改的位置和程度决定了染色质的状态, 影响染色质结构和控制通路的分子配合物, 允许转录活化的7。考虑到核密度和组蛋白修饰在基因转录的局部控制中起着作用, 我们开发了一种本机芯片方法, 可以同时测量核密度和组蛋白修饰9, 10。
本机芯片-seq 利用切 micrococci 核酸 (MNase) 在细胞核内的原生状态中消化完整的染色质11,12, 该属性已被杠杆映射核定位13,14,15. 核密度芯片-seq (ndChIP) 利用 MNase 对染色质的开放区域的优先访问性的特性来生成将 MNase 可访问性与组蛋白修饰10相结合的测量。ndChIP-seq 适用于在罕见的原代细胞, 组织和培养细胞的组蛋白修饰的分析。在这里, 我们提出了一个详细的协议, 使序列数据集的生成适合于先前描述的分析框架工作10 , 它集成了片段大小后沉淀, 由配对端读取边界确定, 以同时调查 MNase 的可访问性与组蛋白修饰测量。此前, 本议定书适用于1万个主要的人脐血衍生 CD34+细胞和人类胚胎干细胞揭示染色质结构和组蛋白修饰之间的独特关系在这些细胞类型中10.考虑到它能够同时测量核的可访问性和组蛋白修饰, ndChIP-seq 能够在单个核级别的单元格中揭示表特征, 并将异构签名解析为组成元素。通过 ndChIP-seq 研究异质细胞群的一个例子是对二价促进剂的调查, 其中两个 H3K4me3, 一个活跃标记, 和 H3K27me3, 一个镇压标记, 存在10。

<table><tbody><tr><td>1M Tris-HCl &amp;ndash;pH 7.5</td><td>Thermo Scientific</td><td>15567-027</td><td></td></tr><tr><td>1M Tris-HCl &amp;ndash;pH 8</td><td>Thermo Scientific</td><td>15568-025</td><td></td></tr><tr><td>0.5M EDTA</td><td>Thermo Scientific</td><td>AM9260G</td><td></td></tr><tr><td>5M NaCl</td><td>Sigma</td><td>1001385276</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>1001412354</td><td></td></tr><tr><td>Sodium-Deoxycholate</td><td>Sigma</td><td>1001437582</td><td></td></tr><tr><td>SDS</td><td>Thermo Scientific</td><td>15525-017</td><td></td></tr><tr><td>Sodium-Bicarbonate</td><td>Fisher Scientific</td><td>S233-500</td><td></td></tr><tr><td>100% EtOH</td><td>NA</td><td>NA</td><td></td></tr><tr><td>V-bottom 96 well plate (AB 1400)</td><td>Thermo Scientific</td><td>AB-1400-L</td><td></td></tr><tr><td>Gilson P2 pipetman</td><td>Mandel</td><td>F144801</td><td></td></tr><tr><td>Gilson P10 pipetman</td><td>Mandel</td><td>F144802</td><td></td></tr><tr><td>Gilson P20 pipetman</td><td>Mandel</td><td>F123600</td><td></td></tr><tr><td>Gilson P100 pipetman</td><td>Mandel</td><td>F123615</td><td></td></tr><tr><td>Gilson P200 pipetman</td><td>Mandel</td><td>F123601</td><td></td></tr><tr><td>Gilson P1000 pipetman</td><td>Mandel</td><td>F123603</td><td></td></tr><tr><td>Micrococcal Nuclease&amp;nbsp;</td><td>NEB</td><td>M0247S</td><td></td></tr><tr><td>Thermo Mixer C (Heating block mixer)</td><td>Eppendorf</td><td>5382000023</td><td></td></tr><tr><td>Multi 12-channel Pippet P20</td><td>Rainin</td><td>17013803</td><td></td></tr><tr><td>Multi 12-channel Pippet P200</td><td>Rainin</td><td>17013805</td><td></td></tr><tr><td>1.5 ml LoBind tube</td><td>Eppendorf</td><td>22431021</td><td></td></tr><tr><td>0.5 ml LoBind tube</td><td>Eppendorf</td><td>22431005</td><td></td></tr><tr><td>Plastic Plate Cover</td><td>Edge Bio</td><td>48461</td><td></td></tr><tr><td>PCR cover</td><td>Bio Rad</td><td>MSD-1001</td><td></td></tr><tr><td>Aluminum Plate Cover</td><td>Bio Rad</td><td>MSF-1001</td><td></td></tr><tr><td>Elution buffer (EB buffer)</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>1M DTT</td><td>Sigma</td><td>646563</td><td></td></tr><tr><td>Eppendorf 5810R centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>22625004</td><td></td></tr><tr><td>Ultrapure water</td><td>Thermo Scientific</td><td>10977-015</td><td></td></tr><tr><td>Protein G dynabeads</td><td>Thermo Scientific</td><td>10001D</td><td></td></tr><tr><td>Protein A dynabeads</td><td>Thermo Scientific</td><td>10001D</td><td></td></tr><tr><td>Protease inhibitor Cocktail</td><td>Calbiochem</td><td>539134</td><td></td></tr><tr><td>Rotating platform</td><td>Mandel</td><td>1b109-12052010</td><td></td></tr><tr><td>Plate magnet</td><td>Alpaqua</td><td>2523</td><td></td></tr><tr><td>Domed 12-cap strip</td><td>Bio Rad</td><td>TCS1201</td><td></td></tr><tr><td>Buffer G2</td><td>Qiagen</td><td>1014636</td><td></td></tr><tr><td>Protease</td><td>Qiagen</td><td>19155</td><td></td></tr><tr><td>Tube Magnet (DynaMag-2)</td><td>Thermo Scientific</td><td>12321D</td><td></td></tr><tr><td>Tube Magnet (DynaMag-2)</td><td>LabCore</td><td>730-006</td><td></td></tr><tr><td>V shape Plastic reservoir</td><td>Mandel</td><td>S0100A</td><td></td></tr><tr><td>Vortex</td><td>Mandel</td><td>C1801</td><td></td></tr><tr><td>Mini Fuge</td><td>Mettler Toledo</td><td>XS2035</td><td></td></tr><tr><td>Analytical scale</td><td>Mandel</td><td>LB109-12052010</td><td></td></tr><tr><td>Quick Ligation Reaction Buffer, 5X</td><td>NEB</td><td>B6058S</td><td></td></tr><tr><td>DNA Polymerase I, Large (Klenow) Fragment</td><td>NEB</td><td>M0210S</td><td></td></tr><tr><td>Klenow Fragment (3&#039;-5&#039; exo)</td><td>NEB</td><td>M0212S</td><td></td></tr><tr><td>T4 DNA Polymerase</td><td>NEB</td><td>M0203S</td><td></td></tr><tr><td>T4 Polynucleotide Kinase</td><td>NEB</td><td>M0201S</td><td></td></tr><tr><td>Deoxynucleotide Solution mix, 10mM</td><td>NEB</td><td>N0447S</td><td></td></tr><tr><td>dATP Solution 10mM</td><td>NEB</td><td>N0440S</td><td></td></tr><tr><td>Quick T4 DNA Ligase</td><td>NEB</td><td>M0202T</td><td></td></tr><tr><td>Adenosine 5&#039;-Triphosphate (ATP), 25mM</td><td>NEB</td><td>P0756S</td><td></td></tr><tr><td>DNA Polymerase</td><td>Thermo Scientific</td><td>F549L</td><td></td></tr><tr><td>NEBuffer 2</td><td>NEB</td><td>B7002S</td><td></td></tr><tr><td>Hank&#039;s buffered salt solution</td><td>Thermo Scientific</td><td>14025076</td><td></td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Thermo Scientific</td><td>A3160702</td><td></td></tr><tr><td>phosphate buffer saline&amp;nbsp;</td><td>Thermo Scientific</td><td>10010023</td><td></td></tr><tr><td>H3K4me3</td><td>Cell Signaling</td><td>9751S</td><td></td></tr><tr><td>H3K4me1</td><td>Diagenode</td><td>C15410037</td><td></td></tr><tr><td>H3K27me3</td><td>Diagenode</td><td>pAb-069-050</td><td></td></tr><tr><td>H3K36me3</td><td>Abcam</td><td>ab9050</td><td></td></tr><tr><td>H3K9me3</td><td>Diagenode</td><td>C15410056</td><td></td></tr><tr><td>SeraMag&amp;nbsp; super-paramagnetic beads</td><td></td><td></td><td></td></tr></tbody></table>

generation of native chromatin immunoprecipitation sequencing libraries for nucleosome density analysis

我们提出了一个改进的本地染色质沉淀测序 (芯片-seq) 实验协议兼容的高斯混合分布的分析方法 (核密度芯片序列; ndChIP-seq), 使生成的micrococcal 核酸 (MNase) 可及性与组蛋白修饰的联合测量。核位置和局部密度, 以及对其组蛋白亚基的后修饰, 协同调节局部转录状态。组合测量的核可达性与组蛋白修饰产生的 ndChIP-seq 允许同时审讯这些功能。ndChIP-seq 方法适用于基于交联的芯片-seq 协议的小数目的原电池。在一起, ndChIP-seq 使测量组蛋白修饰结合局部核密度, 以获得新的洞察力的共享机制, 调节 RNA 转录在稀有的原细胞群体。

Watch this Scientific Journal Video about Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis at JoVE.com

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

我们提出了一个改良的本地染色质沉淀测序 (芯片-seq) 的方法, 以生成序列数据集适合核密度芯片-seq 分析框架集成 micrococcal 核酸 (MNase) 可及性组蛋白修饰测量。

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

generation-native-chromatin-immunoprecipitation-sequencing-libraries

Research

JoVE Journal

Genetics

22.0K 次观看.  University of British Columbia. 我们提出了一个改良的本地染色质沉淀测序 (芯片-seq) 的方法, 以生成序列数据集适合核密度芯片-seq 分析框架集成 micrococcal 核酸 (MNase) 可及性组蛋白修饰测量。

视频: Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

低丰度胚胎标本的染色质沉淀 (芯片) 协议

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

科研

遗传学

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.

Here, we describe an optimized high-throughput ChIP-sequencing protocol and computational analyses pipeline for the determination of genome-wide chromatin state patterns from frozen tumor tissues and cell lines.

肿瘤组织中高通量芯片测序的染色质状态全基因组图谱集成平台

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of ...

癌症研究

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

小鼠棕色脂肪组织的染色质免疫沉淀

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

神经科学

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

生物学

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

该ChroP方法结合芯片和质谱剖析染色质的基因座特异性蛋白质组学风景

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Methods for mapping in vivo protein-DNA interactions are becoming crucial for every aspect of genomic research but they are laborious, costly, and time consuming. Here a commercially available robotic liquid handling system that automates chromatin immunoprecipitation for mapping in vivo protein-DNA interactions with limited amounts of cells is presented.

自动化芯片起实验产生上万HeLa细胞表观遗传配置文件

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

用于大规模表观遗传学研究的半自动ChIP-Seq程序

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&amp;Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single ...

Chromatin Immunoprecipitation to Identify Target Protein Binding Sites on Genomic DNA

Source: Dong, X., et.al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/57547">Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin Immunoprecipitation Sequencing Analysis.</a>&#160;J. Vis. Exp.&#160;(2018)
In this video, we demonstrate the chromatin immunoprecipitation technique to identify protein binding sites on specific regions of the genomic DNA of oligodendrocyte precursor cells via the selective immunoprecipitation of protein-bound chromatin fragments. This method helps to study the interaction of several regulatory proteins, including transcription factors involved in gene regulation.

染色质免疫沉淀鉴定基因组 DNA 上的靶蛋白结合位点

Source: Dong, X., et.al., Identifying Transcription Factor Olig2 Genomic Binding Sites in Acutely Purified PDGFR&#945;+ Cells by Low-cell Chromatin ...