我们感谢哈维尔实验室的其他成员对手稿的反馈。我们感谢CERCA计划，加泰罗尼亚将军和Josep Carreras基金会的机构支持。这项工作由FEDER/西班牙科学与创新部（RTI2018-094788-A-I00），欧洲血液学协会（4823998）和西班牙抗癌协会（AECC）LABAE21981JAVI资助。BMJ由La Caixa银行基金会青年领袖项目（LCF/BQ/PI19/11690001）资助，LR由AGAUR FI奖学金（2019FI-B00017）资助，LT-D由FPI奖学金（PRE2019-088005）资助。我们感谢巴塞罗那自治大学的生物化学和分子生物学博士课程的支持。没有一个资助者在实验设计或手稿写作的任何时候都参与其中。

研究稀缺细胞中启动子相互作用组的综合方法

作者没有什么可透露的。

liCHi-C提供了使用PCHi-C的类似实验框架生成高分辨率启动子相互作用组库的能力，但细胞数量大大减少。通过消除不必要的步骤，例如苯酚纯化和生物素去除，可以极大地实现这一点。在经典的核内连接Hi-C方案57 及其随后的衍生技术PCHi-C中，生物素从非连接的限制性内切性片段中去除，以避免拉下随后没有信息的DNA片段。跳过这部分及其随后的DNA纯化不会显着降低有效读数的百?...

时间基因表达驱动细胞分化，并最终推动生物体发育，其改变与多种疾病密切相关1，2，3，4，5。基因转录受调控元件作用的精细调控，调控元件可分为近端（即基因启动子）和远端（例如增强子或消音器），后者通常位于远离其靶基因的位置，并通过染色质环与它们发生物理相互作用以调节基因表达6，7，8。
基因组中远端调控区域的鉴定是一个被广泛同意的问题，因为这些区域具有特定的组蛋白修饰9，10，11并且包含特定的转录因子识别基序，作为它们的招募平台12，13，14。此外，在增强子和超级增强子15，16的情况下，它们还具有低核小体占用率17，18，并转录为非编码eRNA19，20。
尽管如此，每个远端调控元件的靶基因更难预测。通常情况下，远端调控元件与其靶标之间的相互作用是细胞类型和刺激特异性的21，22，跨越数百千碱基，在任何方向上桥接其他基因23，24，25，甚至可以位于其靶基因或其他非干预基因的内含子区域内26，27.此外，远端调控元件也可以同时控制多个基因，反之亦然28，29。这种位置复杂性阻碍了它们之间的调控关联，因此，每种细胞类型中每个调控元件的大多数靶标仍然是未知的。
近年来，用于研究染色质相互作用的染色体构象捕获（3C）技术的发展出现了显着的繁荣。其中使用最广泛的Hi-C允许生成细胞基因组每个片段之间所有相互作用的图谱30。然而，为了检测限制性片段水平上的显着相互作用，Hi-C依赖于超深度测序，禁止将其用于常规研究单个基因的调控环境。为了克服这种经济限制，已经出现了几种基于富集的3C技术，例如ChIA-PET31，HiChIP 32及其低输入对应物HiCuT33。这些技术依赖于使用抗体来富集由特定蛋白质介导的全基因组相互作用。尽管如此，这些3C技术的独特之处也是其应用的祸根;用户依赖于目标蛋白质的高质量抗体的可用性，并且无法比较蛋白质结合是动态的条件。
启动子捕获Hi-C（PCHi-C）是另一种基于富集的3C技术，可规避这些限制34，35。通过采用生物素化RNA探针富集系统，PCHi-C能够生成全基因组高分辨率基因组区域文库，这些文库与28，650个人类或27，595个小鼠注释的基因启动子（也称为启动子相互作用组）相互作用。这种方法允许人们在活性和非活性启动子的限制性片段水平分辨率下检测显着的长程相互作用，并可靠地比较任何条件之间的启动子相互作用组，而与组蛋白修饰或蛋白质结合的动力学无关。近年来，PCHi-C已被广泛用于鉴定细胞分化过程中的启动子相互作用组重组36，37，鉴定转录因子38，39的作用机制，并发现通过非编码变异40，41，42，43，44，45在疾病中失调的新潜在基因和途径，46，47，48，以及新的驱动程序非编码突变49，50。此外，通过仅修改捕获系统，该技术可以根据生物学问题进行定制，以询问任何相互作用组（例如，增强子相互作用组51或非编码改变的相互作用组41，52）。
然而，PCHi-C依赖于至少2000万个细胞来执行该技术，这阻止了对稀缺细胞群的研究，例如经常用于发育生物学和临床应用的细胞群。出于这个原因，我们开发了低输入捕获Hi-C（liCHi-C），这是一种基于PCHi-C实验框架的具有成本效益和可定制的新方法，用于生成具有低细胞输入的高分辨率启动子相互作用组。通过以最少的试管更换进行实验，交换或消除原始PCHi-C方案中的步骤，大幅减少反应体积并修改试剂浓度，文库复杂性最大化，并且可以生成低至50，000个细胞的高质量文库53。
低输入捕获Hi-C（liCHi-C）已针对PCHi-C进行基准测试，并用于阐明人类造血细胞分化过程中启动子相互作用组重新布线，发现潜在的新疾病相关基因和因非编码改变而失调的途径，并检测染色体异常53。这里详细介绍了分步协议和通过该技术的不同质量控制，直到库的最终生成及其计算分析。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 mM Biotin-14-dATP</td>
			<td>Invitrogen</td>
			<td>19524-016</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris pH 8.0</td>
			<td>Invitrogen</td>
			<td>AM9855G</td>
			<td></td>
		</tr>
		<tr>
			<td>10x NEBuffer 2</td>
			<td>New England Biolabs</td>
			<td>B7002S</td>
			<td>Referenced as restriction buffer 2 in the manuscript</td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Fisher Scientific</td>
			<td>BP3994</td>
			<td></td>
		</tr>
		<tr>
			<td>10x T4 DNA ligase reaction buffer</td>
			<td>New England Biolabs</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde solution (w/v), methanol-free</td>
			<td>Thermo Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mg/mL Bovine Serum Albumin</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Invitrogen</td>
			<td>AM9760G</td>
			<td></td>
		</tr>
		<tr>
			<td>5PRIME Phase Lock Gel Light tubes</td>
			<td>Qiuantabio</td>
			<td>2302820</td>
			<td>For phenol-chloroform purification in section 4 (DNA purification). Phase Lock Gel tubes are a commercial type of tubes specially designed to maximize DNA recovery after phenol-chloroform purifications while avoiding carryover of contaminants in the organic phase by containing a resin of intermediate density which settles between the organic and aqueous phase and isolates them. PLG tubes should be spun at 12,000 x g for 30 s before use to ensure that the resin is well-placed at the bottom of the tube</td>
		</tr>
		<tr>
			<td>Adapters and PCR primers for library amplification</td>
			<td>Integrated DNA Technologies</td>
			<td>-</td>
			<td>Bought as individual primers with PAGE purification for NGS</td>
		</tr>
		<tr>
			<td>Cell scrappers</td>
			<td>Nunc</td>
			<td>179693</td>
			<td>Or any other brand</td>
		</tr>
		<tr>
			<td>Centrifuge (fixed-angle rotor for 1.5 mL tubes)</td>
			<td>Any brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CHiCAGO R package</td>
			<td>1.14.0</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CleanNGS beads</td>
			<td>CleanNA</td>
			<td>CNGS-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>dATP, dCTP, dGTP, dTTP</td>
			<td>Promega</td>
			<td>U120A, U121A, U122A, U123A</td>
			<td>Or any other brand</td>
		</tr>
		<tr>
			<td>DNA LoBind tube, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>30108051</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA LoBind tube, 2 mL</td>
			<td>Eppendorf</td>
			<td>30108078</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA polymerase I large (Klenow) fragment 5000 units/mL</td>
			<td>New England Biolabs</td>
			<td>M0210L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin C1 beads</td>
			<td>Invitrogen</td>
			<td>65002</td>
			<td>For biotin pull-down of the pre-captured library in section 8 (biotin pull-down)</td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin T1 beads</td>
			<td>Invitrogen</td>
			<td>65602</td>
			<td>For biotin pull-down of the post-captured library in section 11 (biotin pull-down and PCR amplification)</td>
		</tr>
		<tr>
			<td>DynaMag-2</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td>Or any other magnet suitable for 1.5 ml tubeL</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR</td>
			<td>20821.321</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS, qualified</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Or any other brand</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Fisher BioReagents</td>
			<td>BP381-1</td>
			<td></td>
		</tr>
		<tr>
			<td>GlycoBlue Coprecipitant</td>
			<td>Invitrogen</td>
			<td>AM9515</td>
			<td>Used for DNA coprecipitation in section 4 (DNA purification)</td>
		</tr>
		<tr>
			<td>HiCUP</td>
			<td>0.8.2</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HindIII, 100 U/&#181;L</td>
			<td>New England Biolabs</td>
			<td>R0104T</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630</td>
			<td>Sigma-Aldrich</td>
			<td>I8896-50ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow EXO- 5000 units/mL</td>
			<td>New England Biolabs</td>
			<td>M0212L</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-retention filter tips&#160;(10 &#181;L, 20 &#181;L, 200 &#181;L and 1000 &#181;L)</td>
			<td>ZeroTip</td>
			<td>PMT233010, PMT252020, PMT231200, PMT252000</td>
			<td></td>
		</tr>
		<tr>
			<td>M220 Focused-ultrasonicator</td>
			<td>Covaris</td>
			<td>500295</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro TUBE AFA Fiber Pre-slit snap cap 6 x 16 mm vials</td>
			<td>Covaris</td>
			<td>520045</td>
			<td>For sonication in section 6 (sonication)</td>
		</tr>
		<tr>
			<td>NheI-HF, 100 U/&#181;L</td>
			<td>New England Biolabs</td>
			<td>R3131M</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free molecular biology grade water</td>
			<td>Sigma-Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers for quality controls</td>
			<td>Integrated DNA Technologies</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR strips&#160;and caps</td>
			<td>Agilent Technologies</td>
			<td>410022, 401425</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol: Chloroform: Isoamyl Alcohol 25:24:1, Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>P3803</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion&#160;High-Fidelity PCR Master Mix with HF Buffer</td>
			<td>New England Biolabs</td>
			<td>M0531L</td>
			<td>For amplification of the library in sections 9 (dATP-tailing, adapter ligation and PCR amplification) 
			and 11 (biotin pull-down and PCR amplification)</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail (EDTA-free)</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR grade</td>
			<td>Roche</td>
			<td>3115836001</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 1x dsDNA High Sensitivity kit</td>
			<td>Invitrogen</td>
			<td>Q33230</td>
			<td>For DNA quantification after precipitation in section 4 (DNA purification)</td>
		</tr>
		<tr>
			<td>Qubit assay tubes</td>
			<td>Invitrogen</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>rCutsmart buffer</td>
			<td>New England Biolabs</td>
			<td>B6004S</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI Medium 1640 1x + GlutaMAX</td>
			<td>Gibco</td>
			<td>61870-010</td>
			<td>Or any other brand</td>
		</tr>
		<tr>
			<td>SDS - Solution 10% for molecular biology</td>
			<td>PanReac AppliChem</td>
			<td>A0676</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate pH 5.2</td>
			<td>Sigma-Aldrich</td>
			<td>S7899-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SureSelect custom 3-5.9 Mb library</td>
			<td>Agilent Technologies</td>
			<td>5190-4831</td>
			<td>Custom designed mouse or human capture system, used for the capture</td>
		</tr>
		<tr>
			<td>SureSelect Target Enrichment Box 1</td>
			<td>Agilent Technologies</td>
			<td>5190-8645</td>
			<td>Used for the capture</td>
		</tr>
		<tr>
			<td>SureSelect Target Enrichment Kit ILM PE Full Adapter</td>
			<td>Agilent Technologies</td>
			<td>931107</td>
			<td>Used for the capture</td>
		</tr>
		<tr>
			<td>T4 DNA ligase 1 U/&#181;L</td>
			<td>Invitrogen</td>
			<td>15224025</td>
			<td>For ligation in section 3 (ligation and decrosslink)</td>
		</tr>
		<tr>
			<td>T4 DNA ligase 2000000/mL</td>
			<td>New England Biolabs</td>
			<td>M0202T</td>
			<td>For ligation in section 9 (dATP-tailing, adapter ligation and PCR amplification)</td>
		</tr>
		<tr>
			<td>T4 DNA polymerase 3000 units/mL</td>
			<td>New England Biolabs</td>
			<td>M0203L</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK 10000 units/mL</td>
			<td>New England Biolabs</td>
			<td>M0201L</td>
			<td></td>
		</tr>
		<tr>
			<td>Tapestation 4200 instrument</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>For automated electrophoresis in section 9 (dATP-tailing, adapter ligation, and PCR amplification) and 
			section 11 (Biotin pull-down and PCR amplification). Any other automated electrophoresis system is valid</td>
		</tr>
		<tr>
			<td>Tapestation reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-5582, 5067-5583, 5067-5584, 5067-5585,</td>
			<td>For automated electrophoresis in section 9 (dATP-tailing, adapter ligation, and PCR amplification) and 
			section 11 (Biotin pull-down and PCR amplification). Any other automated electrophoresis system is valid</td>
		</tr>
		<tr>
			<td>Triton X-100 for molecular biology</td>
			<td>PanReac AppliChem</td>
			<td>A4975</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

an integrated workflow to study the promoter-centric spatio-temporal genome architecture in scarce cell populations

时空基因转录受到远端调控元件（如增强子和消音器）的严格调控，这些元件依赖于与靶基因启动子的物理接近来控制转录。虽然这些调控元件很容易识别，但它们的靶基因很难预测，因为它们中的大多数是细胞类型特异性的，并且可能在线性基因组序列中被数百个千碱基分开，跳过其他非靶基因。几年来，启动子捕获Hi-C（PCHi-C）一直是远端调控元件与其靶基因关联的黄金标准。然而，PCHi-C依赖于数百万个细胞的可用性，禁止研究稀有细胞群，例如通常从原代组织获得的细胞群。为了克服这一限制，已经开发了低输入捕获Hi-C（liCHi-C），这是一种经济高效且可定制的方法来识别控制基因组每个基因的远端调控元件库。liCHi-C依赖于与PCHi-C类似的实验和计算框架，但通过采用最小的试管更换，修改试剂浓度和体积，以及交换或消除步骤，它可以在文库构建过程中实现最小的材料损失。总的来说，liCHi-C能够在发育生物学和细胞功能的背景下研究基因调控和时空基因组组织。

Temporal gene expression drives cell differentiation and, ultimately, organism development, and its alteration is closely related to a wide plethora of diseases1,2,3,4,5. Gene transcription is finely regulated by the action of regulatory elements, which can be classified as proximal (i.e., gene promoters) and distal (e.g., enhancers or silencers), the latter of which are frequently located afar from their target genes and physically interact with them through chromatin looping to modulate gene expression6,7,8.
The identification of distal regulatory regions in the genome is a matter which is widely agreed upon, since these regions harbor specific histone modifications9,10,11 and contain specific transcription factor recognition motifs, acting as recruiting platforms for them12,13,14. Besides, in the case of enhancers and super-enhancers15,16, they also have low-nucleosome occupancy17,18 and are transcribed into non-coding eRNAs19,20.
Nonetheless, each distal regulatory element&#39;s target genes are more difficult to predict. More often than not, interactions between distal regulatory elements and their targets are cell-type and stimulus specific21,22, span hundreds of kilobases, bridging over other genes in any direction23,24,25, and can even be located inside intronic regions of their target gene or other non-intervening genes26,27. Furthermore, distal regulatory elements can also control more than one gene at the same time, and vice versa28,29. This positional complexity hinders pinpointing regulatory associations between them, and therefore, most of each regulatory element&#39;s targets in every cell type remain unknown.
During recent years, there has been a significant boom in the development of chromosome conformation capture (3C) techniques for studying chromatin interactions. The most widely used of them, Hi-C, allows to generate a map of all the interactions between every fragment of a cell&#39;s genome30. However, to detect significant interactions at the restriction fragment level, Hi-C relies on ultra-deep sequencing, prohibiting its use to routinely study the regulatory landscape of individual genes. To overcome this economic limitation, several enrichment-based 3C techniques have emerged, such as ChIA-PET31, HiChIP32, and its low-input counterpart HiCuT33. These techniques depend on the use of antibodies to enrich for genome-wide interactions mediated by a specific protein. Nonetheless, the unique feature of these 3C techniques is also the bane of their application; users count on the availability of high-quality antibodies for the protein of interest and cannot compare conditions in which the binding of the protein is dynamic.
Promoter Capture Hi-C (PCHi-C) is another enrichment-based 3C technique that circumvents these limitations34,35. By employing a biotinylated RNA probe enrichment system, PCHi-C is able to generate genome-wide high-resolution libraries of genomic regions interacting with 28,650 human- or 27,595 mouse-annotated gene promoters, also known as the promoter interactome. This approach allows one to detect significant long-range interactions at the restriction fragment level resolution of both active and inactive promoters, and robustly compare promoter interactomes between any condition independently of the dynamics of histone modifications or protein binding. PCHi-C has been widely used over recent years to identify promoter interactome reorganizations during cell differentiation36,37, identify the mechanism of action of transcription factors38,39, and discover new potential genes and pathways deregulated in disease by non-coding variants40,41,42,43,44,45,46,47,48, alongside new driver non-coding mutations49,50. Besides, by just modifying the capture system, this technique can be customized according to the biological question to interrogate any interactome (e.g., the enhancer interactome51 or the interactome of a collection of non-coding alterations41,52).
However, PCHi-C relies on a minimum of 20 million cells to perform the technique, which prevents the study of scarce cell populations such as the ones often used in developmental biology and clinical applications. For this reason, we have developed low input Capture Hi-C (liCHi-C), a new cost-effective and customizable method based on the experimental framework of PCHi-C to generate high-resolution promoter interactomes with low-cell input. By performing the experiment with minimal tube changes, swapping or eliminating steps from the original PCHi-C protocol, drastically reducing reaction volumes, and modifying reagent concentrations, library complexity is maximized and it is possible to generate high-quality libraries with as little as 50,000 cells53.
Low input Capture Hi-C (liCHi-C) has been benchmarked against PCHi-C and used to elucidate promoter interactome rewiring during human hematopoietic cell differentiation, discover potential new disease-associated genes and pathways deregulated by non-coding alterations, and detect chromosomal abnormalities53. The step-by-step protocol and the different quality controls through the technique are detailed here until the final generation of the libraries and their computational analysis.

作者聚焦：研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程  

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

With liCHi-C, the main question that we are trying to answer is how the chromatin interacts in the nucleus, and therefore, to understand a new layer of gene regulation. We have proven with liCHi-C that we can explore the genome organization of a given sample, link the non-coding mutation associated with disease with potential genes affected, and detect chromosomal rearrangements, such as translocation, for example, in tumor samples. Now we can explore the 3D chromatin organization of scar cell types, such as primary stem cells or relevant clinical samples.Our protocol reduces the input materials and the time and cost needed to obtain quality data to explore the genome organization at the restriction fragment resolution. liCHi-C is expanding the reach of the 3D chromatin organization field, allowing us to explore new cell types previously impossible to analyze. Thanks to liCHi-C, the scientific community can explore the special regulation of gene expression.For example, in malignant transformation only in a specific clonal sub-population inside a tumor.

liCHi-C提供了生成高质量和分辨率的全基因组启动子相互作用组文库的可能性，只需50，000个细胞53。这是通过 - 除了大幅减少反应体积和在整个方案中使用DNA低结合塑料器皿 - 从原始方案中消除不必要的步骤来实现的，在这些步骤中会发生显着的材料损失。这些包括脱钩后的苯酚纯化，生物素去除以及随后的苯酚-氯仿纯化和乙醇沉淀。除此之外，重新组织Hi-C文库制备的步骤（生...

Watch this Scientific Journal Video about An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations at JoVE.com

基因表达由基因启动子与远端调控元件的相互作用调节。在这里，我们解释了低输入捕获Hi-C（liCHi-C）如何允许在稀有细胞类型中鉴定这些相互作用，这些相互作用以前是无法测量的。

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

对于liCHi-C，我们试图回答的主要问题是染色质如何在细胞核中相互作用，从而了解新的基因调控层。我们已经用liCHi-C证明，我们可以探索给定样本的基因组组织，将与疾病相关的非编码突变与潜在的受影响基因联系起来，并检测染色体重排，例如易位，例如肿瘤样本中的易位。现在我们可以探索疤痕细胞类型的3D染色质组织，例如原代干细胞或相关临床样本。我们的方案减少了输入材料以及获得高质量数据以探索限制性片段分辨率的基因组组织所需的时间和成本。liCHi-C正在扩大3D染色质组织领域的覆盖范围，使我们能够探索以前无法分析的新细胞类型。多亏了liCHi-C，科学界可以探索基因表达的特殊调控。例如，仅在肿瘤内的特定克隆亚群中进行恶性转化。

an-integrated-workflow-to-study-promoter-centric-spatio-temporal

Research

JoVE Journal

Genetics

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

968 次观看.  Josep Carreras Leukaemia Research Institute. 对于liCHi-C，我们试图回答的主要问题是染色质如何在细胞核中相互作用，从而了解新的基因调控层。我们已经用liCHi-C证明，我们可以探索给定样本的基因组组织，将与疾病相关的非编码突变与潜在的受影响基因联系起来，并检测染色体重排，例如易位，例如肿瘤样本中的易位。现在我们可以探索疤痕细胞类型的3D染色质组织，例如原代干细胞或相关临床样本。我们的方...

视频: 作者聚焦：研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程  

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

科研

遗传学

Lysis and Digestion of Scarce Cells

To begin, take an aliquot of frozen scarce cells and add 1 milliliter of cold lysis buffer to disrupt the cell membrane. Incubate the cells on ice for 30 minutes, mixing by inversion every five minutes. Centrifuge the nuclei for 10 minutes at 1, 000 G.Remove the supernatant and re-suspend the nuclei in 500 microliters of cold 1.25X restriction buffer 2.Then add 5.5 microliters of 10%sodium dodecyl sulfate, mix by vortexing and incubate in a thermo block. Then quench the SDS by adding 37.5 microliters of 10%Triton X-100. Mix and incubate the sample as demonstrated earlier.Add 7.5 microliters of Hind III to digest the chromatin mix the solution and incubate overnight at 37 degrees Celsius.

稀缺细胞的裂解和消化

Ligation and Decrosslinking of Scarce Cell DNA

To begin, take the digested scarce cell's DNA and place it on ice. Prepare a master mix by using the reagents required to fill in and biotinylate the restriction fragment overhangs. Incubate the sample at 37 degrees Celsius for 75 minutes, mixing by inversion every 15 minutes.Chill the sample on ice, and ligate the filled-in DNA ends by preparing a master mix using the reagents required for ligation. Finally, incubate the sample for four to six hours at 16 degrees Celsius, and then for 30 minutes at room temperature. Add 30 microliters of 10 milligrams per milliliter of Proteinase K to decrosslink the chromatin.Mix the solution, and incubate overnight at 65 degrees Celsius.

稀缺细胞DNA的连接和去交联

Sonication and Biotin Pull-Down of Scarce Cells DNA

After purifying the DNA, begin by taking ligated and decross-linked digested chromatin of the scarce cells. Set the water bath sonicator at a duty factor of 20%peak incidence power of 50 to 200 cycles per burst for 65 seconds, and a temperature range of 6 to 10 degrees Celsius, and sonicate the ligated and decross-linked digested chromatin. After end repairing the sample, transfer 150 microliters of C1 streptavidin beads per sample to a 1.5 milliliter tube.Place them on a 1.5 milliliter tube magnet and wait until all the beads are stuck to the wall. Then remove the supernatant, leaving the beads behind. Next, wash the beads by adding 400 microliters of Tween buffer and resuspending them with soft vortexing.Place the tube back in the magnet until all the beads are stuck to the wall. Then remove the supernatant, leaving the beads behind. After resuspending the beads, in 300 microliters of 2X No-Tween buffer or NTB, combine them with the 300 microliters of the sample.Finally, incubate for 15 minutes rotating at room temperature to pull down the informative DNA fragments with biotin.

稀缺细胞DNA的超声处理和生物素下拉

dATP-Tailing, Adapter Ligation, and PCR Amplification of Scarce Cell DNA

DATP-Tailing, Adapter Ligation, and PCR Amplification of Scarce Cell DNA  

To begin, take the scarce cell's DNA fragments pulled with biotin. Prepare a master mix by adding the reagents for dATP tailing and incubate the sample for 30 minutes at 37 degrees Celsius. Then, inactivate Klenow exo-minus by incubating the sample for 10 minutes at 65 degrees Celsius and cooling it on ice.After washing the beads with 300 microliters each of TB, NTB, and 100 microliters of ligation buffer, resuspend them in 50 microliters of ligation buffer. Add four microliters of 15 micromolar pre-annealed adapter mix and one microliter of 2000 units per microliter T4 DNA ligase to the sample. Wash the beads with 400 microliters of TB, 200 microliters of NTB, and 100 microliters of restriction buffer two.After amplifying the library by PCR, pool all the 50 microliter reactions from the same sample into a 1.5 milliliter DNA low binding tube. Place it on a tube magnet and wait until all the beads are stuck to the wall. Transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube and top up with TLE buffer to 500 microliters.To perform a double-sided selection using paramagnetic bead purification, add 200 microliters of stock beads to the library, mix by vortexing, and incubate for 10 minutes, rotating at room temperature. Then, place the library on a magnet and wait until all the beads are stuck to the wall. Transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube.Concentrate the beads by taking 750 microliters of stock beads and placing them in a 1.5 milliliter tube on a magnet. Once all the beads are stuck to the wall, remove the supernatant and resuspend the beads by vortexing in 300 microliters of new stock beads. Add 300 microliters of concentrated beads to the sample.Mix by vortexing and incubate for 10 minutes, rotating at room temperature. Place on a magnet. Wait until all the beads are stuck to the wall and remove the supernatant.Wash the beads by adding one milliliter of 70%ethanol to the tube with the beads still on the magnet. Allow the beads to air dry and resuspend them in 21 microliters of TLE buffer by vortexing. To elute the library from the beads, incubate the sample for 10 minutes at 37 degrees Celsius in a thermoblock.Place the tube in a magnet and transfer the supernatant containing the library to a new 1.5 milliliter DNA low binding tube.

稀缺细胞DNA的dATP-拖尾、接头连接和PCR扩增

Library Capture, Biotin Pull-Down, and PCR Amplification Using Scarce Cell DNA Library

Begin by taking 500 to 1000 nanograms of the scarce cell DNA library. Dry the DNA with a vacuum concentrator and resuspended it in 3.4 microliters of nuclease-free water. After adding the blockers, and while on the thermocycler, at 65 degrees Celsius, transfer the hybridization solution with the biotinylated RNA to the blocked library.After closing the tube lid firmly, incubate in the thermocycler overnight at 65 degrees Celsius. Then transfer 50 microliters of T1 Streptavidin beads per sample to a 1.5 milliliter DNA low binding tube and place them on a 1.5 milliliter tube magnet. Once all the beads are stuck to the wall, remove the supernatant leaving the beads behind.Now wash the beads thrice with 200 microliters of the binding buffer from the target enrichment kit and resuspend the beads in 200 microliters of binding buffer. Transfer the sample to the resuspended beads while on the thermocycler and incubate for 30 minutes. After washing the beads with 200 microliters of wash buffer 1, incubate them for 15 minutes, rotating at room temperature.Then wash the beads thrice with 200 microliters of wash buffer 2 heated to 65 degrees Celsius and incubate in a thermo block. Finally, after amplifying the library by PCR, perform a DNA purification using paramagnetic beads by adding 270 microliters of stock beads to the sample and mixing by vortexing. An automated electrophoresis profile from a library, just before capture, provided 49.7 nanograms per microliter of DNA in 20 microliters reaction volume.However, 3.06 nanograms per microliter of DNA is obtained from a high sensitivity automated electrophoresis profile from a finished leachic library.