Name: single-cell factor localization on chromatin using ultra-low input cleavage under targets and release using nuclease
Uploaded: 2022-02-01T15:00:00
Description: Watch this Scientific Journal Video about Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease at JoVE.com

我们感谢海纳实验室的成员阅读和评论本手稿的早期版本。该项目使用匹兹堡 UPMC 儿童医院匹兹堡大学健康科学测序核心提供的 NextSeq500 进行测序，特别感谢其主任 William MacDonald。这项研究得到了匹兹堡大学研究计算中心通过提供的计算机资源提供的部分支持。这项工作得到了美国国立卫生研究院拨款号R35GM133732（S.J.H.）的支持。

伦理声明：所有研究均获得匹兹堡大学研究保护机构生物安全办公室的批准。
1. 准备磁珠
注意：在细胞分选前进行，并保持在冰上直到使用。
<ol><li>每次反应移液30μLConA共轭顺磁微球珠浆混合物到新鲜的1.5mL微量离心管中，并加入850μL结合缓冲液，轻轻移液以混合。 注意：共轭顺磁微球是凝集素包被的磁珠，允许脂质膜结合。</li>
	<li>将...

<ol>
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C., Bock, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIPmentation:+fast,+robust,+low-input+ChIP-seq+for+histones+and+transcription+factors.">ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors.</a> Nature Methods. 12 (10), 963-965 (2015).</li><li>Cao, Z., Chen, C., He, B., Tan, K., Lu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+device+for+epigenomic+profiling+using+100+cells.">A microfluidic device for epigenomic profiling using 100 cells.</a> Nature Methods. 12 (10), 959-962 (2015).</li><li>Shankaranarayanan, P., 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=Single-tube+linear+DNA+amplification+(LinDA)+for+robust+ChIP-seq.">Single-tube linear DNA amplification (LinDA) for robust ChIP-seq.</a> Nature Methods. 8 (7), 565-567 (2011).</li><li>Rotem, A., 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=Single-cell+ChIP-seq+reveals+cell+subpopulations+defined+by+chromatin+state.">Single-cell ChIP-seq reveals cell subpopulations defined by chromatin state.</a> Nature Biotechnology. 33 (11), 1165-1172 (2015).</li><li>Grosselin, 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=High-throughput+single-cell+ChIP-seq+identifies+heterogeneity+of+chromatin+states+in+breast+cancer.">High-throughput single-cell ChIP-seq identifies heterogeneity of chromatin states in breast cancer.</a> Nature Genetics. 51 (6), 1060-1066 (2019).</li><li>Adli, M., Bernstein, B. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cChIP-seq:+a+robust+small-scale+method+for+investigation+of+histone+modifications.">cChIP-seq: a robust small-scale method for investigation of histone modifications.</a> BMC Genomics. 16, 1083 (2015).</li><li>Brind'Amour, 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=An+ultra-low-input+native+ChIP-seq+protocol+for+genome-wide+profiling+of+rare+cell+populations.">An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations.</a> Nature Communications. 6, 6033 (2015).</li><li>Schmid, M., Durussel, T., Laemmli, U. K. C. h. I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChlC+and+ChEC;+genomic+mapping+of+chromatin+proteins.">ChlC and ChEC; genomic mapping of chromatin proteins.</a> Molecular Cell. 16 (1), 147-157 (2004).</li><li>Zentner, G. E., Kasinathan, S., Xin, B., Rohs, R., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChEC-seq+kinetics+discriminates+transcription+factor+binding+sites+by+DNA+sequence+and+shape+in+vivo.">ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.</a> Nature Communications. 6, 8733 (2015).</li><li>Skene, P. J., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+targeted+nuclease+strategy+for+high-resolution+mapping+of+DNA+binding+sites.">An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.</a> eLife. 6, 21856 (2017).</li><li>Meers, M. P., Bryson, T. D., Henikoff, J. G., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+CUT&amp;RUN+chromatin+profiling+tools.">Improved CUT&amp;RUN chromatin profiling tools.</a> eLife. 8, 46314 (2019).</li><li>Hainer, S. J., Bo&#353;kovi&#263;, A., McCannell, K. N., Rando, O. J., Fazzio, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+pluripotency+factors+in+single+cells+and+early+embryos.">Profiling of pluripotency factors in single cells and early embryos.</a> Cell. 177 (5), 1319-1329 (2019).</li><li>Kaya-Okur, H. S., 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=CUT&amp;Tag+for+efficient+epigenomic+profiling+of+small+samples+and+single+cells.">CUT&amp;Tag for efficient epigenomic profiling of small samples and single cells.</a> Nature Communications. 10 (1), 1930 (2019).</li><li>Carter, B., 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=Mapping+histone+modifications+in+low+cell+number+and+single+cells+using+antibody-guided+chromatin+tagmentation+(ACT-seq).">Mapping histone modifications in low cell number and single cells using antibody-guided chromatin tagmentation (ACT-seq).</a> Nature Communications. 10 (1), 3747 (2019).</li><li>Gopalan, S., Wang, Y., Harper, N. W., Garber, M., Fazzio, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+profiling+of+multiple+chromatin+proteins+in+the+same+cells.">Simultaneous profiling of multiple chromatin proteins in the same cells.</a> Molecular Cell. 81 (22), 4736-4746 (2021).</li><li>Patty, B. J., Hainer, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+chromatin+profiling+genome-wide+using+uliCUT&amp;RUN+in+single+cells+and+individual+blastocysts.">Transcription factor chromatin profiling genome-wide using uliCUT&amp;RUN in single cells and individual blastocysts.</a> Nature Protocols. 16 (5), 2633-2666 (2021).</li><li>Zheng, X. -. Y., Gehring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-input+chromatin+profiling+in+Arabidopsis+endosperm+using+CUT&amp;RUN.">Low-input chromatin profiling in Arabidopsis endosperm using CUT&amp;RUN.</a> Plant Reproduction. 32 (1), 63-75 (2019).</li><li>Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+native+chromatin+for+fast+and+sensitive+epigenomic+profiling+of+open+chromatin,+DNA-binding+proteins+and+nucleosome+position.">Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.</a> Nature Methods. 10 (12), 1213-1218 (2013).</li><li>Buenrostro, J. 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CUT&RUN是确定染色质上蛋白质定位的有效方案。与其他协议相比，它具有许多优点，包括：1）高信噪比，2）快速协议，以及3）所需的低排序读取覆盖率，从而节省了成本。使用蛋白A-或蛋白A / G-MNase使CUT&RUN能够与任何可用的抗体一起使用;因此，它有可能快速，轻松地分析许多蛋白质。然而，对于染色质上的任何蛋白质分析，适应单细胞是困难的，特别是与单细胞转录组学（即scRNA-seq）相比，由于...

许多核蛋白通过与染色质相互作用来促进或阻止DNA模板化活性。为了确定这些染色质相互作用蛋白的功能，重要的是要确定这些蛋白结合的基因组位置。自1985年开发以来，染色质免疫沉淀（ChIP）一直是鉴定蛋白质与染色质1，2结合的黄金标准。传统的ChIP技术具有以下基本工作流程：收获细胞并交联（通常与甲醛），剪切染色质（通常使用苛刻的超声处理方法，需要交联），使用靶向蛋白质（或标记蛋白质）的抗体免疫沉淀，然后使用二抗（与琼脂糖或磁珠偶联），交联被逆转， 蛋白质和RNA被消化以纯化DNA，并且该ChIP富集DNA被用作分析模板（使用放射性标记的探针1，2，qPCR3，微阵列5，6或测序4）。随着微阵列和大规模并行深度测序的出现，ChIP-chip5，6和ChIP-seq4最近被开发出来，并允许全基因组鉴定染色质上的蛋白质定位。交联ChIP自问世以来一直是一种强大而可靠的技术，ChIP-exo7和ChIP-nexus8在分辨率方面取得了重大进展。在开发 ChIP-seq 的同时，已经建立了用于 ChIP （N-ChIP） 的天然（非交联）方案，其利用核酸酶消化（通常使用微球菌核酸酶或 MNase）来片段化染色质，而不是在传统的交联 ChIP 技术中进行的超声处理9.然而，交联ChIP和N-ChIP技术的一个主要缺点是由于实验操作后的DNA产量低，因此对高细胞数量的需求。因此，近年来，许多努力都是为了优化ChIP技术以实现低电池输入。这些努力导致开发了许多强大的基于ChIP的技术，这些技术的适用性和输入要求10，11，12，13，14，15，16，17，18各不相同。然而，基于单细胞ChIP-seq的技术一直缺乏，特别是对于非组蛋白。
2004年，开发了一种替代技术来确定染色质上的蛋白质占用率，称为染色质内源性裂解（ChEC）和染色质免疫裂解（ChIC）19。这些单位点技术利用MNase与目标蛋白（ChEC）或蛋白A（ChIC）的融合，以直接切割与目标蛋白相邻的DNA。近年来，ChEC和ChIC都针对染色质的全基因组蛋白质分析进行了优化（分别为ChEC-seq和CUT&RUN）20，21。虽然ChEC-seq是确定因子定位的强大技术，但它需要为每个靶标开发MNase融合蛋白，而ChIC及其全基因组变异CUT&RUN依赖于针对目标蛋白（如ChIP）和重组蛋白A-MNase的抗体，其中蛋白A可以识别抗体的IgG恒定区域。作为替代方案，已经开发了融合蛋白A /蛋白G-MNase（pA / G-MNase），可以识别更广泛的抗体常数区域22。CUT&RUN已迅速成为ChIP-seq的流行替代品，用于确定染色质全基因组上的蛋白质定位。
超低输入CUT&RUN（uliCUT&RUN）是CUT&RUN的一种变体，可以使用低和单单元输入，在2019年进行了描述23。这里，描述了手动96孔格式单细胞应用的方法。值得注意的是，自uliCUT&RUN开发以来，已经开发了组蛋白分析的两种替代方案，CUT&Tag和iACT-seq，为组蛋白24，25提供了稳健且高度平行的分析。此外，scCUT&Tag已经过优化，可以分析单个细胞中的多种因子（multiCUT&Tag）并应用于非组蛋白26。总之，CUT&RUN为低输入ChIP-seq提供了一种有吸引力的替代方案，其中uliCUT&RUN可以在任何可以使用细胞分选机和标准设备的分子生物学实验室中进行。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL clear microfuge tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>90410</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube magnetic rack</td>
			<td>ThermoFisher Scientific</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube-compatible cold centrifuge</td>
			<td>Eppendorf</td>
			<td>5404000537</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm sterile tissue culture plates</td>
			<td>ThermoFisher Scientific</td>
			<td>150464</td>
			<td></td>
		</tr>
		<tr>
			<td>10X T4 DNA Ligase buffer</td>
			<td>New England Biolabs</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>VWR</td>
			<td>89039-656</td>
			<td></td>
		</tr>
		<tr>
			<td>1X TE buffer</td>
			<td>ThermoFisher Scientific</td>
			<td>12090015</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L PCR tubes</td>
			<td>Eppendorf</td>
			<td>951010022</td>
			<td></td>
		</tr>
		<tr>
			<td>2X quick ligase buffer</td>
			<td>New England Biolabs</td>
			<td>M2200</td>
			<td>Ligase Buffer</td>
		</tr>
		<tr>
			<td>5X KAPA HiFi buffer</td>
			<td>Roche</td>
			<td>7958889001</td>
			<td>5X high fidelity PCR buffer</td>
		</tr>
		<tr>
			<td>7-Amino-Actinomycin D (7-AAD)</td>
			<td>Fisher Scientific</td>
			<td>BDB559925</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well magnetic rack</td>
			<td>ThermoFisher Scientific</td>
			<td>12027 or 12331D</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>VWR</td>
			<td>82006-636</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>polystyrene-magnetite beads; Due to potential variability between AMPure XP bead lots, it is recommended that your AMPure bead solution be calibrated. See manufacturer&#8217;s instructions</td>
		</tr>
		<tr>
			<td>Antibody to protein of interest</td>
			<td>varies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>ThermoFisher Scientific</td>
			<td>R0441</td>
			<td></td>
		</tr>
		<tr>
			<td>BioMag Plus Concanavalin A beads</td>
			<td>Polysciences</td>
			<td>86057-10</td>
			<td>ConA-conjugated paramagnetic microspheres</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2)</td>
			<td>Fisher Scientific</td>
			<td>AAJ62905AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>BD FACSAria II cell sorter</td>
			<td></td>
			<td>Requires training</td>
		</tr>
		<tr>
			<td>Cell-specific media for cell culture</td>
			<td>Varies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>ThermoFisher Scientific</td>
			<td>C298-500</td>
			<td>Chloroform is a skin irritant and harmful if swallowed; handle in a chemical fume hood using gloves, a lab coat, and goggles</td>
		</tr>
		<tr>
			<td>Computer with 64-bit processer and access to a super computing cluster</td>
			<td></td>
			<td></td>
			<td>For computational analyses of resulting sequencing datasets</td>
		</tr>
		<tr>
			<td>DNA spin columns</td>
			<td>Epoch Life Sciences</td>
			<td>1920-250</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP set</td>
			<td>New England Biolabs</td>
			<td>N0446S</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis equipment</td>
			<td>varies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>22032601</td>
			<td>100% vol/vol ethanol is highly flammable; handle in a chemical fume hood using gloves, a lab coat, and goggles</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid&#160; (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>BP2482100</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>VWR</td>
			<td>97063-256</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Heterologous S. cerevisiae DNA spike-in</td>
			<td>homemade</td>
			<td></td>
			<td>Prepared from crosslinked, MNase-digested, and agarose gel extracted genomic DNA purified of protein/RNA and diluted to 10 ng/mL. We recommend yeast genomic DNA, but other organisms can be used if needed.</td>
		</tr>
		<tr>
			<td>Hydrochloric Acid&#160; (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144-212</td>
			<td>Hydrochloric Acid is very corrosive; handle in a chemical fume hood using gloves, a lab coat, and goggles</td>
		</tr>
		<tr>
			<td>Ice Bucket</td>
			<td>varies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina Sequencing platform (e.g., NextSeq500)</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator with temperature and atmosphere control</td>
			<td>ThermoFisher Scientific</td>
			<td>51030284</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA HotStart HiFi DNA Polymerase with 5X KAPA HiFi buffer</td>
			<td>Roche</td>
			<td>7958889001</td>
			<td>hotstart high fidelity polymerase</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Bakery Company</td>
			<td>SG404</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese Chloride (MnCl2)</td>
			<td>Sigma Aldrich</td>
			<td>244589</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette set</td>
			<td>Rainin</td>
			<td>30386597</td>
			<td></td>
		</tr>
		<tr>
			<td>Minifuge</td>
			<td>Benchmark Scientific</td>
			<td>C1012</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB Adaptor</td>
			<td>New England Biolabs</td>
			<td>E6612AVIAL</td>
			<td>Adaptor</td>
		</tr>
		<tr>
			<td>NEB Universal primer</td>
			<td>New England Biolabs</td>
			<td>E6611AVIAL</td>
			<td>Universal Primer</td>
		</tr>
		<tr>
			<td>NEBNext Multiplex Oligos for Illumina kit</td>
			<td>New England Biolabs</td>
			<td>E7335S/L, E7500S/L, E7710S/L, E7730S/L</td>
			<td>Indexed Primers</td>
		</tr>
		<tr>
			<td>Negative control antibody</td>
			<td>Antibodies-Online</td>
			<td>ABIN101961</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease Free water</td>
			<td>New England Biolabs</td>
			<td>B1500S</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR thermocycler</td>
			<td>Eppendorf</td>
			<td>2231000666</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase lock tubes</td>
			<td>Qiagen</td>
			<td>129046</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol/Chloroform/Isoamyl Alcohol (PCI)</td>
			<td>ThermoFisher Scientific</td>
			<td>15593049</td>
			<td>Phenol is harmful if swallowed or upon skin contact; handle in a chemical fume hood using gloves, a lab coat, and goggles</td>
		</tr>
		<tr>
			<td>Phsophate buffered saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>10814010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette aid</td>
			<td>Drummond Scientific</td>
			<td># 4-000-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene glycol (PEG) 4000</td>
			<td>VWR</td>
			<td>A16151</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Fisher Scientific</td>
			<td>P250-1</td>
			<td>CAUTION KOH is an eye/skin irritant as a solid and corrosive in solution. Handle in a chemical fume hood using gloves, a lab coat, and goggles</td>
		</tr>
		<tr>
			<td>Protease Inhibitors</td>
			<td>ThermoFisher Scientific</td>
			<td>78430</td>
			<td></td>
		</tr>
		<tr>
			<td>ProteinA/G-MNase</td>
			<td>Epicypher</td>
			<td>15-1016</td>
			<td>pA/G-MNase</td>
		</tr>
		<tr>
			<td>ProteinA-MNase, purified from pK19pA-MN</td>
			<td>Addgene</td>
			<td>86973</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>New England Biolabs</td>
			<td>P8107S</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 1X dsDNA HS Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>Q33230</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Fluorometer</td>
			<td>ThermoFisher Scientific</td>
			<td>Q33238</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Ligase with 2X Quick Ligase buffer</td>
			<td>New England Biolabs</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>New England Biolabs</td>
			<td>T3010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate&#160; (NaOAc)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP333-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S5150-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP166-500</td>
			<td>SDS is poisonous if inhaled; handle with care in well ventilated spaces using gloves, eye protection, and an N95-grade respirator when handling</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>S318-1</td>
			<td>NaOH is an eye/skin irritant as a solid and corrosive in solution. Handle in a chemical fume hood using gloves, a lab coat, and goggles</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma Aldrich</td>
			<td>S2626</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Inverted Light Microscope</td>
			<td>Leica</td>
			<td>11526213</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard lab agarose gel materials</td>
			<td>Varies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard lab materials such as serological pipettes and pipette tips</td>
			<td>Varies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0203S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop vortexer</td>
			<td>Fisher Scientific</td>
			<td>2215414</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0273S</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>5384000020</td>
			<td>Alternatively, can use a waterbath</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>9002-93-1</td>
			<td>Triton X-100 is hazardous; use a lab coat, gloves, and goggles when handling</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Fisher Scientific</td>
			<td>MT25052</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube rotator</td>
			<td>VWR</td>
			<td>10136084</td>
			<td></td>
		</tr>
		<tr>
			<td>USER enzyme</td>
			<td>New England Biolabs</td>
			<td>M5505S</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell factor localization on chromatin using ultra-low input cleavage under targets and release using nuclease

确定蛋白质在染色质上的结合位置对于了解其功能和潜在的调节靶点至关重要。30多年来，染色质免疫沉淀（ChIP）一直是确定蛋白质定位的黄金标准，其定义是使用抗体从超声处理或酶消化染色质中提取出感兴趣的蛋白质。最近，由于抗体系留技术增加了敏感性，因此在评估染色质上的蛋白质定位方面变得流行起来。靶标下的裂解和核酸酶下的释放（CUT&RUN）是染色质免疫裂解（ChIC）的全基因组衍生物，并利用与微球菌核酸酶（pA-MNase）相连的重组蛋白A来鉴定靶向目标蛋白的抗体的IgG恒定区域，从而实现靶向目标蛋白侧面的DNA的位点特异性裂解。CUT&RUN可用于分析组蛋白修饰，转录因子和其他染色质结合蛋白，如核小体重塑因子。重要的是，CUT&RUN可用于评估真色素或异色相关蛋白质和组蛋白修饰的定位。由于这些原因，CUT&RUN是确定各种蛋白质结合谱的强大方法。最近，CUT&RUN已经针对低细胞群和单细胞的转录因子分析进行了优化，优化的方案被称为超低输入CUT&RUN（uliCUT&RUN）。在这里，以手动96孔格式提出了使用uliCUT&RUN进行单细胞因子分析的详细方案。

在这里，提出了一个详细的方案，用于使用96孔手动格式uliCUT&RUN对染色质进行单细胞蛋白质分析。虽然结果会根据所分析的蛋白质（由于蛋白质丰度和抗体质量），细胞类型和其他影响因素而有所不同，但本文将讨论该技术的预期结果。细胞质量（细胞外观和活细胞百分比）和单细胞分选应在活细胞分选到NE缓冲液之前或期间进行评估。ES细胞集落和细胞分选的示例如图2A<str...

Watch this Scientific Journal Video about Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease at JoVE.com

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

CUT&RUN及其变体可用于确定染色质上的蛋白质占用率。该协议描述了如何使用单细胞uliCUT&RUN确定染色质上的蛋白质定位。

使用靶标下的超低输入切割在染色质上的单细胞因子定位，并使用核酸酶释放

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin ...

CUT&RUN是一种确定染色质上蛋白质定位的强大技术，在这里我们以手动96孔格式描述了该技术的单细胞版本。大多数蛋白质定位技术，如ChIP-seq，需要高细胞输入，大约需要三天才能完成。CUT&RUN由于具有高信号和低背景，已针对低电池和单电池应用进行了优化，并且可以在一天内完成。我们设想该技术几乎可以应用于任何生物样品，并且可以特别有效地确定稀有和低丰度样品（例如珍贵患者样品）中的因子定位。在进行任何单细胞实验之前，请在大量非珍贵细胞上测试抗体和技术。该实验的主要局限性是对强效抗体的依赖。证明该程序的是桑塔纳·拉多（Santana Lardo），他是我实验室经验丰富的研究专家。将单个细胞分选并将细胞核结合到磁珠上后，将96孔板放在96孔磁架上，让磁珠结合至少五分钟。然后，取出并丢弃上清液。向细胞核结合的微球中加入100微升封闭缓冲液，与温和移液混合，并在室温下孵育五分钟。将板放在96孔磁性架上，让上清液清除至少五分钟。然后，除去并丢弃上清液而不干扰磁珠。从磁性架上取下板，并在每次反应中将磁珠重悬于100微升洗涤缓冲液中，轻轻移液。将板放回96孔磁性架上，让上清液清除，然后取出并丢弃上清液。每次反应将微球重悬于25微升洗涤缓冲液中，轻轻移液。通过每次反应等分25微升洗涤缓冲液，然后每次反应加入0.5微升抗体来制作一抗主混合物。加入25.5微升抗体洗涤缓冲液混合物，然后轻轻移液到用靶向目标蛋白质的抗体处理的每个样品上。在室温下孵育一小时。然后，再次将样品放回96孔磁性架上。一旦上清液清除，将其取出并丢弃，而不会干扰珠子。从磁性架上取下板后，用100微升洗涤缓冲液洗涤珠子，并通过移液重悬。将板放回96孔磁性架上并如上所述丢弃上清液后，将每个样品重悬于25微升洗涤缓冲液中。通过每次反应加入25微升洗涤缓冲液和优化量的蛋白质A MNase来制造蛋白质A MNase主混合物。向每个样品（包括对照样品）中加入25微升蛋白质A MNase主混合物。将样品在室温下孵育30分钟。将板放在 96 孔磁性支架上。让上清液清除至少五分钟，然后除去并丢弃上清液而不干扰磁珠。从磁性架上取出板后，用100微升洗涤缓冲液洗涤珠子，并通过轻柔移液重悬。将板放在96孔磁性架上，并如前所述丢弃上清液。然后，从磁性架上取出样品，并通过轻柔移液将磁珠重悬于50微升洗涤缓冲液中。将样品在冰水混合物中平衡至零摄氏度五分钟，然后将样品从冰水浴中取出。使用多通道移液器加入一微升100毫摩尔氯化钙。使用大容量多通道移液器轻轻移液，充分混合三到五次，然后将样品返回零摄氏度。一旦板回到冰水浴中，就启动10分钟的计时器。通过将含有两倍浓度的RSTOP +和缓冲液的溶液移液到每个孔中以与加入氯化钙相同的顺序移液50微升来停止反应。将样品在37摄氏度下孵育20分钟。在四摄氏度下以16， 000倍G旋转板五分钟。将板放在 96 孔磁性支架上。让上清液清除至少五分钟。然后，将上清液转移到新鲜的96孔板中并丢弃磁珠。对于DNA提取，向每个样品中加入一微升10%SDS和0.83微升每毫升蛋白酶K20毫克，并通过温和移液混合样品。将样品在70摄氏度下孵育10分钟。将板恢复到室温。加入46.6微升五摩尔氯化钠和90微升50%PEG4000，轻轻移液混合。向每个样品中加入33微升聚苯乙烯磁铁矿珠，并在室温下孵育10分钟。将板放在磁性架上，让上清液清除约五分钟。然后，小心地丢弃上清液而不干扰珠子。用150微升80%乙醇冲洗两次，而不会干扰珠子。以1000倍G短暂旋转平板30秒。将板放回96孔磁性架上，并在不干扰磁珠的情况下除去所有残留的乙醇。将样品风干约两到五分钟。将磁珠重悬于37.5微升pH 8的10毫摩尔TRIS盐酸盐中，并在室温下孵育5分钟。将板放回磁性架上，让磁珠粘合五分钟。将36.5微升上清液转移到与新鲜热循环仪相容的96孔板中，并丢弃磁珠。在进行细胞质量评估后，检查了细胞外观和百分比，表明不应使用低质量的胚胎干细胞。使用Hoechst 33342染色进行单细胞分选，并对测试细胞进行计数以确保在每个孔中发现零个或一个细胞。琼脂糖凝胶分析揭示了低分子量分子量标和单个单细胞uliCUT&RUN文库。次优和最优文库分析显示低分子量阶梯，由于Mnase消化效率低下而导致的次优文库，以及具有适当消解的成功文库。片段分析仪分布表明，单细胞的预期大小范围为150至500个碱基对。然而，在大蛋白质中，DNA将有大约270个碱基对。使用描述高细胞数或阴性对照的单位点基因组浏览器的蛋白质占用和单细胞CTCF uliCUT&RUN显示，单细胞在很大程度上代表强峰，类似于高细胞uliCUT&RUN。单细胞CTCF或阴性对照的热图揭示了直接在细胞中已建立的CTCF结合位点上进行读富集的清晰模式。一维热图显示，相对于周围区域和无抗体对照，单细胞库中已建立的CTCF结合位点具有更高的读取密度，证明了已建立结合位点的抗体特异性富集。平衡冰水浴上的珠子结合核，并且在添加氯化钙时不使样品过热对于防止染色质过度消化非常重要。

Research

JoVE Journal

Biology

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

采用芯片识别亚型特定的靶基因的全基因组分析

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

科研

生物学

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

成年小鼠心肌细胞的单细胞转录谱

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Chromatin Immunoprecipitation ChIP using Drosophila tissue

染色质免疫沉淀（ChIP）使用果蝇组织

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

纳米有限元分析：蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

生物质的使用限制金额高效的染色质免疫沉淀

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

质粒载体产生表达FLAG标记的蛋白质在人延伸因子1α启动子的使用吉布森大会规则

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

具体的基因组区域及相关分子鉴定enChIP隔离

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

单探头的应用：根据环境条件质谱成像和单细胞分析

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

用数据无关采集质谱法定量测定点特异蛋白赖氨酸乙酰化和琥珀酰化化学计量学

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...