Name: adaptation of hybridization capture of chromatin-associated proteins for proteomics to mammalian cells
Uploaded: 2018-06-01T13:30:00
Description: Watch this Scientific Journal Video about Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells at JoVE.com

这项工作得到了 NIH 资助 P50HG004952 和 R01GM109099 的支持。

1. 捕获寡核苷酸设计
<ol><li>
设计在目标区域/s 的杂交捕获期间使用的寡核苷酸小组。
	<ol>
<li>目的设计每个目标区域的4–8寡核苷酸, 但最低限度, 设计至少一个针对目标区域的每一端的寡核苷酸。</li>
		<li>如果目标序列是长的 (> 500 基对), 设计出尽可能分散的寡核苷酸, 以确保在整个目标区域有效地丰富染色质。 注意: 我们观察到了4–8寡核苷酸的最佳捕获。虽然这个过程可以很好...

<ol>
	<li>Consortium, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2013).</li><li>Galas, D. J., Schmitz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNAse+footprinting:+a+simple+method+for+the+detection+of+protein-DNA+binding+specificity.">DNAse footprinting: a simple method for the detection of protein-DNA binding specificity.</a> Nucleic Acids Res. 5 (9), 3157-3170 (1978).</li><li>Simon, J. M., Giresi, P. G., Davis, I. J., Lieb, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+formaldehyde-assisted+isolation+of+regulatory+elements+(FAIRE)+to+isolate+active+regulatory+DNA.">Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA.</a> Nat. Protoc. 7 (2), 256-267 (2012).</li><li>Dai, Y., Kennedy-Darling, J., Shortreed, M. R., Scalf, M., Gasch, A. P., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Sequence-Specific+Capture+of+Chromatin+and+Mass+Spectrometric+Discovery+of+Associated+Proteins.">Multiplexed Sequence-Specific Capture of Chromatin and Mass Spectrometric Discovery of Associated Proteins.</a> Anal. Chem. 89 (15), 7841-7846 (2017).</li><li>Guillen-Ahlers, H., 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=HyCCAPP+as+a+tool+to+characterize+promoter+DNA-protein+interactions+in+Saccharomyces+cerevisiae.">HyCCAPP as a tool to characterize promoter DNA-protein interactions in Saccharomyces cerevisiae.</a> Genomics. 107 (6), (2016).</li><li>Kennedy-Darling, 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=Discovery+of+Chromatin-Associated+Proteins+via+Sequence-Specific+Capture+and+Mass+Spectrometric+Protein+Identification+in+Saccharomyces+cerevisiae.">Discovery of Chromatin-Associated Proteins via Sequence-Specific Capture and Mass Spectrometric Protein Identification in Saccharomyces cerevisiae.</a> J Proteome Res. 13 (8), 3810-3825 (2014).</li><li>Guillen-Ahlers, H., Shortreed, M. R. R., Smith, L. M. M., Olivier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+methods+for+the+analysis+of+chromatin-associated+proteins.">Advanced methods for the analysis of chromatin-associated proteins.</a> Physiol Genomics. 46 (13), 441-447 (2014).</li><li>Lambert, J. -. P., Fillingham, J., Siahbazi, M., Greenblatt, J., Baetz, K., Figeys, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+budding+yeast+chromatin-associated+interactome.">Defining the budding yeast chromatin-associated interactome.</a> Mol Syst Biol. 6, 448 (2010).</li><li>Soldi, M., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Proteomic+Investigation+of+Chromatin+Functional+Domains+Reveals+Novel+Synergisms+among+Distinct+Heterochromatin+Components.">The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components.</a> Mol Cell Proteomics. 12 (3), 764-780 (2013).</li><li>Wang, C. I., 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=Chromatin+proteins+captured+by+ChIP-mass+spectrometry+are+linked+to+dosage+compensation+in+Drosophila.">Chromatin proteins captured by ChIP-mass spectrometry are linked to dosage compensation in Drosophila.</a> Nat Struct Mol Biol. 20 (2), 202-209 (2013).</li><li>Byrum, S. D., Raman, A., Taverna, S. D., Tackett, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChAP-MS:+A+Method+for+Identification+of+Proteins+and+Histone+Posttranslational+Modifications+at+a+Single+Genomic+Locus.">ChAP-MS: A Method for Identification of Proteins and Histone Posttranslational Modifications at a Single Genomic Locus.</a> Cell Rep. 2 (1), 198-205 (2012).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+specific+genomic+regions+and+identification+of+associated+immunoprecipitation+(+eChIP+)+using+CRISPR.">Efficient isolation of specific genomic regions and identification of associated immunoprecipitation ( eChIP ) using CRISPR.</a> Biochem. Bioph. Res. Co. 439 (1), 132-136 (2013).</li><li>Liu, X., 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=In+Situ+Capture+of+Chromatin+Interactions+by+Biotinylated+dCas9.">In Situ Capture of Chromatin Interactions by Biotinylated dCas9.</a> Cell. 170 (5), 1028-1043 (2017).</li><li>Myers, S. A., Wright, J., Zhang, F., Carr, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-APEX-mediated+proximity+labeling+enables+discovery+of+proteins+associated+with+a+predefined+genomic+locus+in+living+cells.">CRISPR/Cas9-APEX-mediated proximity labeling enables discovery of proteins associated with a predefined genomic locus in living cells.</a> bioRxiv. , (2017).</li><li>D&#233;jardin, J., Kingston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Proteins+Associated+with+Specific+Genomic+Loci.">Purification of Proteins Associated with Specific Genomic Loci.</a> Cell. 136 (1), 175-186 (2009).</li><li>Buxton, K. E., 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=Elucidating+Protein-DNA+Interactions+in+Human+Alphoid+Chromatin+via+Hybridization+Capture+and+Mass+Spectrometry.">Elucidating Protein-DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry.</a> J. Proteome Res. 16 (9), 3433-3442 (2017).</li><li>Kennedy-Darling, J., Holden, M. T., Shortreed, M. R., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+programmable+release+of+captured+DNA.">Multiplexed programmable release of captured DNA.</a> Chembiochem. 15 (16), 2353-2356 (2014).</li><li>Fujita, T., Fujii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+specific+genomic+regions+and+identification+of+associated+molecules+by+engineered+DNA-binding+molecule-mediated+chromatin+immunoprecipitation+(enChIP)+using+CRISPR.">Isolation of specific genomic regions and identification of associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR.</a> Chromatin Protoc. Third Ed. , 43-52 (2015).</li><li>Lambert, J. -. P., Mitchell, L., Rudner, A., Baetz, K., Figeys, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Proteomics+Approach+for+the+Discovery+of+Chromatin-associated+Protein+Networks.">A Novel Proteomics Approach for the Discovery of Chromatin-associated Protein Networks.</a> Mol Cell Proteomics. 8 (4), 870-882 (2009).</li></ol>

这里描述的 HyCCAPP 方法有许多独特的特点, 使它成为一个强有力的方法来揭示 DNA-相互作用, 否则将仍然难以捉摸。这个过程的性质使 HyCCAPP 在基因组的不同有机体和区域工作的灵活性。然而, 这是一个方法, 有几个限制要考虑。
HyCCAPP 是一种避免任何单元格修改的方法, 以便它可以在主细胞、细胞培养系统甚至组织样本中应用。然而, 由于这一原因, 它需要将样品交联, 从而降低...

在过去的十年中, 测序技术有了显著的改进, 允许对大量样本中的各种基因组进行研究, 并具有惊人的分辨率。DNA 元素百科全书 (编码) 联合体是国家卫生研究院国家人类基因组研究所牵头的大规模多机构努力, 它提供了关于个人转录因子如何和其他调控蛋白结合在一起, 并与基因组相互作用。最初的努力的特点是特定的 dna 蛋白相互作用, 由染色质免疫沉淀 (芯片) 评估超过100已知的 dna 结合蛋白 1.替代方法, 如 DNase 足迹2和甲醛辅助隔离的管理元素 (自由放任)3也被用来定位特定区域的基因组相互作用的蛋白质, 但明显的局限性,这些实验方法不能识别相互作用的蛋白质。尽管过去几年进行了广泛的努力, 但没有任何技术能有效地使染色质中蛋白质-DNA 相互作用的综合特征, 以及染色质相关蛋白的鉴定和定量化。
为了解决这一需要, 我们开发了一种新的方法, 我们称之为杂交捕获的染色质相关蛋白的蛋白质组学 (HyCCAPP)。最初开发的酵母4,5,6, 该方法分离的交联染色质区域的利益 (与绑定蛋白) 使用序列特定的杂交捕获。在分离蛋白质-DNA 复合物后, 可以用质谱等方法来表征与兴趣序列有关的蛋白质集。因此, HyCCAPP 可以被认为是一种无偏见的方法来揭示新的 DNA-蛋白质相互作用, 在这个意义上, 它不依赖于抗体, 它是完全不可知的蛋白质, 可能被发现。还有其他方法可以发现新的 DNA 相互作用的蛋白质7, 但大多数依赖于类似芯片的方法8,9,10, 质粒插入11,12, 13、14或具有高拷贝数15的区域。相比之下, HyCCAPP 可以应用于多和单拷贝区域, 并且不需要任何先前关于该区域蛋白质的信息。此外, 虽然上面提到的一些方法有很有价值的特点, 特别是避免了 DNA 蛋白交联反应的需要, 但 HyCCAPP 的独特特点是它可以应用于未修饰细胞中的单拷贝区域, 而且没有任何事先了解假定的结合蛋白, 或可用的抗体。
在这一点上, HyCCAPP 主要用于分析酵母中的不同基因组区域4,5,6, 最近被用于分析α卫星 dna 中的蛋白质-DNA 相互作用, 重复区域在人类基因组16中。作为我们正在进行的工作的一部分, 我们已经适应了最初开发的酵母染色质的杂交捕获方法, 适用于人体细胞的分析, 并提出了一个修改的协议, 允许选择性捕获单拷贝目标人类基因组中的区域, 其效率与我们在酵母中的初步研究相似。这种新的优化协议现在允许技术的适应和利用, 通过质谱或其他分析方法来审问人类基因组中的蛋白质-DNA 相互作用。
重要的是要强调, HyCCAPP 方法是为了分析特定的目标区域, 还不适合于全基因组的分析。这项技术在处理那些缺乏相互作用的蛋白质信息的区域时尤其有用, 或者当需要对特定基因组轨迹上的相互作用蛋白质进行更全面的深入分析时。HyCCAPP 的目的是揭示 dna 结合蛋白, 但不能准确描述基因组 DNA 中特定的蛋白质结合点。在目前的实施中, 该方法并没有提供有关单个蛋白质的 DNA 结合序列或图案的信息。因此, 它很好地补充了现有的技术, 如自由放任, 并可能允许识别新的结合蛋白在基因组区域由初步的自由放任分析确定。

<table>
	<tbody>
		<tr>
			<td>PrimerQuest tool</td>
			<td>IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OligoAnalyzer 3.1</td>
			<td>IDT</td>
			<td></td>
			<td>Analyze capture oligonucleotids</td>
		</tr>
		<tr>
			<td>PairFold</td>
			<td>UBC</td>
			<td></td>
			<td>Analyze interactions</td>
		</tr>
		<tr>
			<td>RPMI 1640 media</td>
			<td>Thermo Fisher</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Thermo Fisher</td>
			<td>25030081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess automated cell counter</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>850 cm2 roller bottle&#160;</td>
			<td>Greiner Bio-one</td>
			<td>680058</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller system</td>
			<td>Wheaton</td>
			<td>22-288-525</td>
			<td></td>
		</tr>
		<tr>
			<td>37 % formaldehyde&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I3021</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P4380</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Thermo Fisher</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Branson digital sonifier SFX 150</td>
			<td>Emerson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 3.0 fluorometer</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR assay kit</td>
			<td>Thermo Fisher</td>
			<td>Q32850</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer 2100</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 kit</td>
			<td>Agilent</td>
			<td>5067-1504</td>
			<td></td>
		</tr>
		<tr>
			<td>MES sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>M3885</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl 5M</td>
			<td>Thermo Fisher</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-2 magnet</td>
			<td>Thermo Fisher</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-15 magnet</td>
			<td>Thermo Fisher</td>
			<td>12301D</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-280 atreptavidin</td>
			<td>Thermo Fisher</td>
			<td>60210</td>
			<td></td>
		</tr>
		<tr>
			<td>Low binding tubes</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>SciGene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tube shaker and rotator</td>
			<td>Thermo Fisher</td>
			<td>415110Q</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I (RNase-free)</td>
			<td>New England BioLabs</td>
			<td>M0303</td>
			<td></td>
		</tr>
		<tr>
			<td>SSC buffer 20&#215; concentrate</td>
			<td>Sigma-Aldrich</td>
			<td>S6639</td>
			<td></td>
		</tr>
	</tbody>
</table>

adaptation of hybridization capture of chromatin-associated proteins for proteomics to mammalian cells

初步开发了用于蛋白质组学 (HyCCAPP) 技术的染色质相关蛋白的杂交捕获, 以揭示酵母中新的 DNA-蛋白质相互作用。它允许对目标区域进行分析, 而不需要事先了解绑定到目标区域的可能蛋白质。理论上, 这使得 HyCCAPP 可以用来分析任何感兴趣的基因组区域, 并且它提供了足够的灵活性, 可以在不同的细胞系统中工作。这种方法并不意味着研究已知转录因子的结合点, 这项任务更适合于染色质免疫沉淀 (芯片) 和类似芯片的方法。HyCCAPP 的力量在于它探索 DNA 区域的能力, 因为它有有限的或不了解与之相关的蛋白质。它也可以是一种方便的方法, 以避免偏见 (目前在芯片样的方法) 引入蛋白质的染色质浓缩使用抗体。潜在的, HyCCAPP 可以是一个强大的工具, 发现真正的新的 DNA 蛋白相互作用。迄今为止, 该技术主要应用于酵母细胞或哺乳动物细胞中的高复制重复序列。为了成为我们设想的强大工具, HyCCAPP 方法需要进行优化, 以有效地捕获哺乳动物细胞中的单拷贝基因座。在这里, 我们提出了我们对人类细胞系的初始酵母 HyCCAPP 捕获协议的适应, 并表明, 单拷贝染色质区域可以有效地与此修改的协议隔离。

由于需要大量的 HyCCAPP 的染色质来获得成功, 细胞生长到相对较高的融合水平。台盼蓝染色用于确认细胞死亡率适中 (< 10%)。在单拷贝实验中, 杂交捕获之前的染色质含量需要在 femtomolar 范围内, 通常需要至少10个9细胞作为起始材料。在进行全面实验之前, 建议在较小的批次中测试捕获寡核苷酸的性能。在图 1中显示了使用寡核苷酸滴定的杂交?...

Watch this Scientific Journal Video about Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells at JoVE.com

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

这是一种方法, 以确定新的 DNA 相互作用的蛋白质在特定的靶点, 依赖序列特定捕获的交联染色质的后续蛋白质组分析。不需要事先了解潜在的结合蛋白, 也无需进行细胞修饰。该技术最初是为酵母开发的, 现在已经适应了哺乳动物细胞。

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

一种快速、定量的后向基因组转化和变异的多肽映射方法

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

CRISPR Guide RNA Cloning for Mammalian Systems

哺乳动物系统 CRISPR 指南 RNA 克隆

The outlined protocol describes streamlined methods for the efficient and cost-effective generation of Cas9-associated guide RNAs. Two alternative ...

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

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

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 ...

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

使用哺乳动物细胞中的微球菌核酸酶进行染色质免疫沉淀测定

To express cellular phenotypes in organisms, living cells execute gene expression accordingly, and transcriptional programs play a central role in gene ...