该芯片外型方法：确定蛋白质-DNA相互作用与近基部精密配对
Summary
Here, we present a protocol to achieve near base pair resolution of protein-DNA interactions. This is obtained by exonuclease treatment of DNA fragments selectively enriched by chromatin immunoprecipitation (ChIP-exo) followed by high throughput sequencing.
Abstract
染色质免疫沉淀（ChIP）是表观遗传学和基因调节的隔离特定蛋白质-DNA相互作用领域不可缺少的工具。沉淀耦合到高通量测序（芯片起）通常用于确定与染色质相互作用的蛋白质的基因组位置。然而，芯片起是由几百个碱基对相对低的映射分辨率和高背景信号受到阻碍。芯片外的方法是芯片起的改良版本对分辨率和噪声大大提高。芯片外方法论的主要区别是：在图书馆准备的工作流程，有效足迹左侧的lambda核酸外切酶消化的引入和蛋白质-DNA交联网站的权5'DNA边界。然后芯片外库进行高通量测序。所得到的数据可以被利用，以提供独特的和超高分辨率分析上市功能组织ÒF中的基因组中。在这里，我们描述了我们优化和精简了哺乳动物系统和新一代测序边合成平台芯片外的方法。
Introduction
染色质免疫沉淀（ChIP）是一种强有力的方法通过对与在活细胞中给定蛋白质相互作用的DNA片段选择性富集来研究基因调控机制。的ChIP富集的DNA片段的检测方法已演变随着技术的改进，从检测单一位点（标准芯片定量PCR）的对寡核苷酸芯片（芯片芯片），以高通量测序（芯片起）1杂交。虽然芯片起已经看到了广泛的应用，染色质异质性和非特异性DNA的相互作用，阻碍了数据质量，导致误报和不准确的映射。为了规避这些限制，弗兰克普格博士开发芯片外方法2。沉淀 – 外的显着特点是，它包括一个5'至3'外切核酸酶，从而有效地足迹转录因子结合的位置。其结果，芯片外的方法实现了更高的分辨率，detectio的更大的动态范围N，和较低的背景噪音。
虽然沉淀外型更加技术上具有挑战性的掌握比芯片起，它被广泛地研究的目的使用不同的生物系统3-8获得独特的超高分辨率的见解采纳。实际上，芯片外已成功地应用于细菌，酵母，小鼠，大鼠和人的细胞系统。作为证据原则，芯片外原本用于识别的精确结合基序的酵母转录一把因素2。也用于在酵母中的技术，研究了转录预起始复合物的组织，并破译各种组蛋白9,10 subnucleosomal结构。最近，我们利用的ChIP-外解决相邻TFIIB和Pol II结合在促进人的事件，并表明广泛转录分歧源于不同的起始复合物11。
这里提出的工作流程最优化和streamlined用于哺乳动物的ChIP -外（ 图1）。首先，住伯或组织培养细胞用甲醛处理通过共价交联的体内蛋白-DNA相互作用保存。将细胞裂解和染色质剪切至〜100 – 500碱基对大小的片段。沉淀然后选择性富集为交联到感兴趣的蛋白质的DNA片段。在这点上，芯片起库通常制备，这固有地限制了检测分辨率到几百个碱基对的平均片段大小。然而，芯片外克服通过调整左和右5所述蛋白质-DNA交联位点以拉姆达核酸外切酶的“DNA边界此限制。测序文库，然后从外切核酸酶消化的DNA构建了如下详述。所得嵌套5'边界代表蛋白质-DNA相互作用（ 图1，步骤14）的体内足迹，并且通过高通量测序进行检测。 Alt键霍夫芯片外的方法比芯片起更多的参与，多数步骤之间的转换需要简单洗珠，最大限度地减少样品损失和实验的可变性。重要的是，由于沉淀外型是芯片起，即成功的芯片起也应该是成功的为片外的任何样品的改良版本。
在从芯片起一个根本不同的数据结构的ChIP外型结果蛋白-DNA相互作用的体内足迹。虽然通用芯片起呼叫者可以应用到芯片外的数据，以获得最精确的峰值呼叫，我们建议专门为片外数据心目中的独特的结构设计，生物信息学工具。这些措施包括Genetrack，创业板，MACE，Peakzilla和ExoProfiler 12-15。
Protocol
Representative Results
Discussion
我们提出了一个功能基因组学的协议，以确定染色质以接近碱基对的分辨率相互作用的蛋白质在一个不带偏见，全基因组的方式精确绑定位置。实现近基部对映射解决最关键的一步就是芯片富集DNA的核酸外切酶处理而免疫沉淀留在磁性树脂。从表面上看，蛋白质复合物有可能阻断体内的任何给定的亚单位（如染色质重塑复合物或核小颗粒）的足迹。然而，如以前报道10中…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank the Venters Lab and members of the Molecular Physiology and Biophysics Department for helpful discussions. Special thanks to Frank Pugh for his guidance, mentoring, and many insightful discussions on the nuances of ChIP-exo while I was a post-doctoral fellow in his laboratory.
Materials
| 37% formaldehyde, methanol free, 10x10ml ampules | ThermoFisher Scientific | 28908 | Section 3 |
| Complete Protease Inhibitor cocktail (CPI) | Roche Life Science | 11873580001 | Sections 4, 8 |
| Bioruptor sonicator | Diagenode | UCD200 | Section 5 |
| 15 ml polystyrene tubes | BD Falcon | 352095 | Section 5 |
| MagSepharose Protein G Xtra beads | GE Healthcare | 28-9670-66 | Section 6 |
| DynaMag-1.5ml Side Magnetic Rack | Invitrogen | 12321D | Sections 6-17, 22 |
| Mini-Tube Rotator | Fisher Scientific | 05-450-127 | Sections 7, 22 |
| T4 DNA Polymerase | New England BioLabs | M0203 | Section 9 |
| DNA Polymerase I, Klenow | New England BioLabs | M0210 | Section 9 |
| 10x NEBuffer 2 (10x Reaction Buffer 2) | New England BioLabs | B7002 | Sections 9-11, 13, 15, 20 |
| Thermomixer C | Eppendorf | 5382 | Sections 9-16 |
| ATP | Roche Life Science | 010419979001 | Sections 9, 11, 13 |
| dNTPs | New England BioLabs | N0447 | Sections 9, 12, 19, 23 |
| T4 Polynucleotide Kinase | New England BioLabs | M0201 | Sections 9, 13 |
| dATP | New England BioLabs | N0440 | Sections 10, 20 |
| Klenow 3'-5' Exo Minus | New England BioLabs | M0212 | Sections 10, 20 |
| T4 DNA ligase | New England BioLabs | M0202 | Sections 11, 21 |
| Φ-29 DNA Polymerase | New England BioLabs | M0269 | Sections 12, 19 |
| 10x Φ-29 Buffer | New England BioLabs | B0269 | Sections 12, 19 |
| Lambda Exonuclease | New England BioLabs | M0262 | Section 14 |
| 10x Lambda Buffer | New England BioLabs | B0262 | Section 14 |
| RecJf Exonuclease | New England BioLabs | M0264 | Section 15 |
| Proteinase K | Roche Life Science | 03115828001 | Section 16 |
| Glycogen | Roche Life Science | 010901393001 | Section 18 |
| 10x T4 Ligase Buffer | New England BioLabs | B0202 | Section 21 |
| AMPure XP (clean up) beads | Beckman Coulter | A63881 | Section 22 |
| Q5 Hot Start DNA Polymerase | New England BioLabs | M0493 | Section 23 |
| 5x Q5 Buffer (5x PCR Buffer) | New England BioLabs | B9027 | Section 23 |
| Qubit Fluorometer | Invitrogen | Q33216 | Section 25 |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | Section 25 |
| Optical Clear Qubit tubes | Invitrogen | Q32856 | Section 26 |
| Qubit dsDNA High Sensitivity Assay kit | Invitrogen | Q32851 | Section 26 |
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