使用色素免疫沉淀-外核酶（ChIP-Exo）对小鼠干细胞衍生神经元中的蛋白质-DNA相互作用进行高分辨率映射
Summary
精确确定整个基因组中的蛋白质结合位置对于理解基因调控非常重要。在这里，我们描述了一种基因组映射方法，它用外核酶消化（ChIP-exo）处理染色质免疫沉淀DNA，然后进行高通量测序。该方法可检测哺乳动物神经元中具有近碱基对映射分辨率和高信号噪声比的蛋白质-DNA相互作用。
Abstract
识别基因组上特定的蛋白质-DNA相互作用对于理解基因调控具有重要意义。色素免疫沉淀与高通量测序（ChIP-seq）广泛用于识别DNA结合蛋白的全基因组结合位置。然而，ChIP-seq方法受其声波DNA片段长度和非特定背景DNA的异质性限制，导致DNA结合位点的制图分辨率低且不确定。为了克服这些限制，ChIP与外核酶消化（ChIP-exo）的结合利用5’到3’外核酶消化来修剪异构大小的免疫沉淀DNA到蛋白质-DNA交叉连接部位。外核酶治疗也消除了非特定背景DNA。图书馆准备的和外核酶消化的DNA可以发送用于高通量测序。ChIP-exo 方法允许近基对映射分辨率，具有更高的检测灵敏度和更小的背景信号。下文描述了哺乳动物细胞和下一代测序的优化 ChIP-exo 协议。
Introduction
蛋白质与DNA相互作用的位置提供了对基因调控机制的洞察。色素免疫沉淀加上高通量测序（ChIP-seq）已经使用了十年来研究活细胞1、2的全基因组蛋白质-DNA相互作用。然而，ChIP-seq方法受DNA片段的异质性和未绑定的DNA污染的限制，导致低映射分辨率、误报、未接听电话和非特定背景信号。ChIP与外核酶消化（ChIP-exo）的结合，通过将ChIPDNA修剪到蛋白质-DNA交叉连接点，提供接近碱基对分辨率和低背景信号3、4、5，从而改进了ChIP-seq方法。ChIP-exo 提供的大大改进的映射分辨率和低背景使整个基因组中能够确定准确和全面的蛋白质-DNA 结合位置。ChIP-exo 能够揭示功能上独特的 DNA 结合图案、转录因子 （TF） 和特定基因组结合位置的多个蛋白质-DNA 交叉链接位点之间的协同相互作用，其他基因组映射方法3、4、6、7无法检测。
ChIP-exo最初用于初露头角的酵母，以检查TF的序列特异性DNA结合，研究转录启动前复合物的精确组织，以及整个基因组4、8、9的单个组酮的亚核体结构。自2011年推出以来，ChIP-exo已成功地用于许多其他生物体，包括细菌、小鼠和人类细胞7、10、11、12、13、14、15、16、17。2016 年，Rhee 等人14首次在哺乳动物神经元中使用 ChIP-exo 来了解在编程 TF Lhx3 降低调节后神经元基因表达是如何维持的，该编程与另一个编程 TF Isl1 形成了一个异质复合体。这项研究表明，在没有Lhx3的情况下，Isl1被招募到受Onecut1 TF约束的新神经元增强剂中，以维持神经元效应基因的基因表达。在这项研究中，ChIP-exo揭示了多个TF如何以接近核苷酸制图分辨率的组合方式动态识别细胞类型和细胞阶段特异性DNA调控元素。其他研究还使用ChIP-exo方法来理解其他哺乳动物细胞系中蛋白质和DNA之间的相互作用。Han等人利用ChIP-exo使用ChIP-exo来研究小鼠红细胞中GATA1和TAL1 TF的全基因组组织。这项研究发现，TAL1是直接招募到DNA，而不是间接通过蛋白质-蛋白质相互作用与GATA1在整个红细胞分化。最近的研究还利用ChIP-exo对CTCF、RNA聚合酶II和平石标记的全基因组结合位置进行剖析，以研究人类细胞系18、19的表观和转录机制。
有几个版本的ChIP-exo协议可用3，5，20。然而，对于不熟悉下一代测序库准备的研究，这些 ChIP-exo 协议很难遵循。ChIP-exo 协议的优秀版本发布时附有易于遵循的说明和视频21，但包含许多酶步骤，需要大量时间才能完成。在这里，我们报告了包含减少酶步骤和潜伏时间的ChIP-exo协议的新版本，以及每个酶步骤21的解释。使用末端预制酶，将末端修复和 dA 尾部反应一步一步地组合在一起。使用夹带增强剂将索引和通用适配器连体步骤的潜伏时间从 2 小时缩短到 15 分钟。删除上一个 ChIP-exo 协议中描述的索引适配器连结步骤后的激酶反应。相反，在寡头DNA合成过程中，磷酸盐组被添加到索引适配器（表1）的5’端之一，这将用于羊田外核酶消化步骤。虽然以前的 ChIP-exo 协议使用 RecJf外核酶消化来消除单链 DNA 污染物，但此消化步骤在这里被删除，因为它对 ChIP-exo 库的质量不至关重要。此外，为了在ChIP分离后净化反向交叉DNA，使用磁珠净化方法代替苯酚：氯仿：异酰醇（PCIA）提取方法。这缩短了DNA提取的潜伏时间。重要的是，它去除索引适配器结对过程中形成的大多数适配器调色器，这可能会影响结对介质 PCR 的效率。
此处提出的ChIP-exo协议进行了优化，用于检测与小鼠胚胎干细胞（ES）细胞分化的哺乳动物神经元的精确蛋白质-DNA相互作用。简言之，收获和交叉链接的神经元细胞被裂解，让色素暴露在声波中，然后进行声波化，从而获得适当大小的DNA片段（图1）。然后，抗体涂层珠子用于有选择地免疫沉淀支离破碎的可溶性色素到感兴趣的蛋白质。当免疫沉淀DNA仍在珠子上时，进行末修复、测序适配器的结对、填充反应和5’至3’lambda外核酶消化步骤。外核酶消化步骤使ChIP-exo具有超高分辨率和高信号噪声比。Lambda 外核酶将免疫沉淀的 DNA 从交叉连接部位修剪出几个碱基对 （bp），从而导致污染的 DNA 降解。外核酶处理的ChIP DNA从抗体涂层珠子中分离出，蛋白质-DNA交叉链路被逆转，蛋白质退化。脱氧核糖核酸被提取并去自然到单链ChIPDNA，然后引物退化和扩展，使双链DNA（dsDNA）。接下来，执行将通用适配器与外核酶处理末端的连结。由此产生的DNA被纯化，然后PCR放大，凝胶纯化，并接受下一代测序。
ChIP-exo 协议比 ChIP-seq 协议长，但在技术上并不十分具有挑战性。任何成功的免疫沉淀的ChIPDNA都可以接受ChIP-exo，并具有几个额外的酶步骤。ChIP-exo在基因组结合位点方面的显著优势，如超高映射分辨率、减少背景信号、减少误报和阴性，超过了时间成本。
Protocol
Representative Results
Discussion
在此协议中，ChIP随后的外核酶消化用于获取DNA库，以超高映射分辨率识别哺乳动物细胞中的蛋白质-DNA相互作用。许多变量有助于ChIP-exo实验的质量。关键的实验参数包括抗体的质量、声波的优化以及LM-PCR周期的数量。这些关键的实验参数也是可以限制ChIP-exo实验，并将在下面讨论。
在任何 ChIP 协议中，抗体质量都是最重要的考虑因素之一。建议使用ChIP级抗体。在执行 ChIP-exo …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢 Rhee 实验室的成员分享未发布的数据和宝贵的讨论。这项工作得到了加拿大自然科学和工程研究理事会（NSERC）赠款RGPIN-2018-06404（H.R.）的支持。
Materials
| Agarose, UltraPure | Invitrogen | 16500 | Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1) |
| Albumin, Bovine Serum (BSA), Protease Free, Heat Shock Isolation, Min. 98% | BioShop | ALB003 | Blocking Solution |
| Antibody against Isl1 | DSHB | 39.3F7 | Antibody incuation with beads (Section 5.6) |
| Bioruptor Pico | Diagenode | B01060010 | Sonicating Chromatin (Section 3.3) |
| Bovine serum albumin (BSA), Molecular Biology Grade | New England BioLabs | B9000S | Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2) |
| Centrifuge 5424 R | Eppendorf | 5404000138 | Sonicating Chromatin (Section 3.5), Checking Sonication (Section 4.3.1, 4.3.3 and 4.3.4) |
| Centrifuge 5804 R | Eppendorf | 22623508 | Harvest, cross-linking and freezing cells (Section 1.3), Cell lysis (Section 2.2 and 2.3), Sonicating Chromatin (Section 3.1) |
| cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Roche | 4693159001 | Added to all buffers, except Proteinase K buffer and ChIP Elution buffer |
| dATP Solution | New England BioLabs | N0440S | dA-Tailing Reaction (Section 15.1) |
| Deoxycholic Acid Sodium Salt | fisher scientific | BP349 | Lysis Buffer 3, High Salt Wash Buffer and LiCl Wash Buffer |
| dNTP Mix, Molecular Biology Grade | Thermo Scientific | R0192 | Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.2) |
| DreamTaq Green PCR Master Mix, 2x | Thermo Scientific | K1081 | Ligation-Mediated PCR (Section 18.1) |
| EDTA, 0.5 M, Sterile Solution, pH 8.0 | BioShop | EDT111 | Lysis Buffer 1-3, Checking Sonication (Section 4.1), High Salt Wash Buffer, LiCl Wash Buffer and ChIP Elution Buffer |
| Ethyl Alcohol Anhydrous, 100% | Commercial alcohols | P006EAAN | Checking Sonication (Section 4.3.3) |
| Formaldehyde, 36.5-38%, contains 10-15% methanol | Sigma | F8775 | Harvest, cross-linking and freezing cells (Section 1.1) |
| Glycerol, Reagent Grade, min 99.5% | BioShop | GLY002 | Lysis Buffer 1 |
| Glycine, Biotechnology Grade, min. 99% | BioShop | GLN001 | Harvest, cross-linking and freezing cells (Section 1.2) |
| Glycogen, RNA Grade | Thermo Scientific | R0551 | Checking Sonication (Section 4.3.3) |
| HEPES, 1 M Sterile-filtered Solution, pH 7.3 | BioShop | HEP003 | Lysis Buffer 1, High Salt Wash Buffer |
| Klenow Fragment (3->5 exo-) | New England BioLabs | M0212S | dA-Tailing Reaction (Section 15.1) |
| Lambda exonuclease | New England BioLabs | M0262S | Lambda Exonuclease Digestion on Beads (Section 11.2) |
| Ligase Enhancer | New England BioLabs | E7645S | NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1) |
| Ligase Master Mix | New England BioLabs | E7645S | NEBNext Ultra II DNA Library Kit. Index Adapter Ligation on Beads (Section 9.1) and Universal Adapter Ligation (Section 16.1) |
| Lithium Chloride (LiCl), Reagent grade | Bioshop | LIT704 | LiCl Wash Buffer |
| Magnetic beads for ChIP (Dynabeads Protein G) | Dynabeads Protein G (magnetic beads for ChIP) | Dynabeads Protein G (magnetic beads for ChIP) | Antibody incubation with beads (Section 5) |
| Magnetic beads for DNA purification (AMPure XP Beads) | Beckman Coulter | A63880 | DNA Extraction (Section 13.3) and DNA Clean-up (Section 17.1) |
| Magnetic rack (DynaMag-2 Magnet) | Invitrogen | 12321D | Used in many steps in Sections: 5 – 11, 13 |
| MinElute Gel Extraction Kit | Qiagen | 28604 | Gel Purification of PCR Amplified DNA (Section 19.2) |
| N-Lauroylsarcosine sodium salt solution, 30% aqueous solution, ≥97.0% (HPLC) | Sigma | 61747 | Lysis Buffer 3 |
| Octylphenol Ethoxylate (IGEPAL CA630) | BioShop | NON999 | Lysis Buffer 1 and LiCl Wash Buffer |
| Phenol:Chloroform:Isoamyl Alcohol, Biotechnology Grade (25:24:1) | BioShop | PHE512 | Checking Sonication (Section 4.3.1) |
| phi29 DNA Polymerase | New England BioLabs | M0269L | Fill-in Reaction on Beads (Section 10.2) and Denaturing, Primer Annealing and Primer Extension (Section 14.3 and 14.4) |
| Phosphate-Buffered Saline (PBS), 1x | Corning | 21040CV | Harvest, cross-linking and freezing cells (Section 1.3) and Sonicating Chromatin (Section 3.1), Antibody incuation with beads (Section 5.1) |
| PowerPac Basic Power Supply | BioRad | 1645050 | Checking Sonication (Section 4.3.6) and Gel Purification of LM-PCR Amplified DNA (Section 19.1) |
| ProFlex PCR System | Applied Biosystems | ProFlex PCR System | Used in Sections: 14.2, 14.4, 15.2, 16.2 and 18.2 |
| Protein LoBind Tube, 2.0 mL | Eppendorf | 22431102 | Antibody Incubation with Beads (Section 5.2) and Chromatin Immunoprecipitation (Section 6.3) |
| Proteinase K Solution, RNA Grade | Invitrogen | 25530049 | Checking Sonication (Section 4.2) and Elution and Reverse Crosslinking (Section 12.3) |
| Qubit 4.0 Fluorometer | Invitrogen | Q33226 | Gel Purification of PCR Amplified DNA (Section 19.3) |
| Quibit dsDNA BR assay kit | Invitrogen | Q32853 | Gel Purification of PCR Amplified DNA (Section 19.3) |
| Rnase A, Dnase and Protease-free | Thermo Scientific | EN0531 | Checking Sonication (Section 4.3.2) and DNA Extraction (Section 13.2) |
| Sodium chloride (NaCl), BioReagent | Sigma | S5886 | Lysis Buffer 1-3, High Salt Wash Buffer |
| Sodium Dodecyl Sulfate (SDS), Electrophoresis Grade | BioShop | SDS001 | Checking Sonication (Section 4.1), High Salt Wash Buffer and ChIP Elution Buffer |
| Sonication beads and 15 mL Bioruptor Tubes | Diagenode | C01020031 | Sonicating Chromatin (Section 3.1 and 3.2) |
| ThermoMixer F1.5 | Eppendorf | 5384000020 | Section 4.2, 4.3.2, 4.3.5, 8.2, 9.2, 10.3, 11.2, 12.2, 12.3, 13.2 and 16.2 |
| Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 7.4 | Sigma | T2194 | 10 mM Tris-HCl Buffer |
| Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 8.0 | Sigma | T2694 | Lysis Buffer 2, Lysis Buffer 3, Checking Sonication (Section 4.1), LiCl Wash Buffer and ChIP Elution Buffer |
| Ultra II End Repair/dA-Tailing Module (24 rxn -> 120 rxn) | New England BioLabs | E7546S | End Prep Reaction mix and End Prep Enzyme mix. End-repair and dA-Tailing Reaction on Beads (Section 8.2) |
| VWR Mini Tube Rocker, Variable Speed | VWR | 10159-752 | Used in many steps in sections: 1, 2, 5, 6 and 7 |
| 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Triton X-100, Reagent Grade | BioShop | TRX506 | Lysis Buffer 1, Sonicating Chromatin (Section 3.4) and High Salt Wash Buffer |
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