tchip-seq: 细胞专用表观体分析
1RNA Biology Laboratory,RIKEN, 2RNA Systems Biochemistry Laboratory,RIKEN Cluster for Pioneering Research, 3Laboratory for Phyloinformatics,RIKEN Center for Biosystems Dynamics Research, 4RNA Biology Laboratory, Faculty of Pharmaceutical Sciences,Hokkaido University, 5Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences,University of Tokyo
Summary
我们描述了串联染色质免疫沉淀测序 (tchip-seq) 的分步协议, 该协议可分析细胞类型特异性基因组蛋白的全组蛋白修饰。
Abstract
表观遗传调控在基因表达中起着核心作用。自20世纪60年代发现组蛋白修饰以来, 其生理和病理功能得到了广泛的研究。事实上, 下一代深度测序和染色质免疫沉淀 (chip) 通过特异性组蛋白修饰抗体的出现, 已经彻底改变了我们对整个基因组表观遗传调控的看法。相反, 组织通常由不同的细胞类型组成, 其复杂的混合物对研究特定细胞类型的表观基因组提出了分析挑战。为了以全基因组的方式解决细胞类型特异性染色质状态, 我们最近开发了串联染色质免疫沉淀测序 (tchip-seq), 它是基于从细胞标记的核心组蛋白纯化染色质的选择类型的兴趣, 其次是 chip-seq。该协议的目标是引入 tchip-seq 的最佳实践。该技术为组织特异性表观基因组研究提供了一种通用工具, 用于不同组蛋白修饰和模型生物的研究。
Introduction
动物的组织由不同的细胞类型组成。每个细胞的基因调控定义了细胞类型。染色质修饰-dna 甲基化和组蛋白修饰-是基因表达的细胞类型特异性的基础。因此, 每种细胞类型的表观遗传调控的测量都是人们所希望的, 但这一直是一个技术挑战。
为了研究特定细胞类型的表观遗传学, 最近开发了串联染色质免疫沉淀测序 (tchip-seq)( 图 1)1。在 tchip 中, 表位标记的核心组蛋白 h2b 是从细胞类型特异性启动子中表达的。此功能允许从感兴趣的细胞中分离色谱素, 尽管这种材料从各种细胞类型的混合物开始。在 chip-seq–通过修饰组蛋白标记和下一代分离 dna 深度测序进行纯化之后, 我们可以以全基因组的方式监测目标细胞类型的表观遗传状态。
利用这项技术, 我们最近研究了在赖氨酸 4 (h3k4me3) 标记组蛋白 h3 蛋白的神经元特异性三甲基化。在这项研究中, 我们开发了一个敲门砖, 其中 c 端结标记的 h2b 蛋白表达后, cre-acid 介导重组 (rosa26cag 浮法 h2b-flag)。在 camk2a 启动子的控制下, 利用具有 cre-内质网 (er) 基因的小鼠杂交, 得到的小鼠线在他莫西芬注射液 (camk2ah2b-flag)1 后诱导活性神经元 h22-flag。从已建立的小鼠线的大脑开始, 我们用抗 h3k4me3 抗体进行了 tchip-seq。由于 h3k4me3 标记通常对应于启动子区域, 我们可以发现在神经元1中特别表达的数百个 mrna。
在这里, 我们描述了一个典型的 tchip-seq 方法, 它涵盖了从组织解剖到库构造的步骤(图 1)。本协议的最终目标是分享我们在 tchip-seq 性能方面的最佳实践, 以及此方法今后在其他单元格类型和组蛋白修饰中的应用。
Protocol
此处描述的所有方法均已获得 riken (h27-ep071) 安全部门的批准, 并遵循相关的准则和法规。 1. 组织解剖 使用精细的弹簧剪刀将感兴趣的组织分解成小块 (约 lt;3 毫米2)。注意: 较大的组织碎片需要更长的时间来冻结, 较小的碎片将携带较大体积的缓冲液, 这两者都可能影响结果。 将解剖的组织碎片加入一个充满液氮的清洁容器中, 收集到2毫升充满液氮的…
Representative Results
在这里, 我们描述了组织解剖, 固定, 细胞裂解, 染色质的串联纯化, 以及 dna 文库准备下一代测序。在过程中, 可以在多个步骤中测试 dna 的质量, 这是成功测序的关键 (图 2)。由于单个核糖体通常被 147 bp dna 4包围, 剪切的 dna 不应短于该大小。超声检查后, 立即分离 dna 并在微流式电泳机上运行 (图 2a)。尽管我们?…
Discussion
我们的协议针对小鼠大脑的神经元进行了优化, 其中 FLAG-tagged 的 h2b 的表达是由他莫西芬注射液诱导的。用于 h2b 表达的促进剂、起始组织材料和组织量是 tchip-seq 成功的关键参数。因此, 应考虑对每个细胞类型感兴趣的因素进行优化。
在本协议中使用的程序中, 一个关键的步骤是 dna 剪切, 以实现 200-500 bp5的染色质长度。一般来说, 超声检查步骤的标准化具有?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢岩崎实验室的所有成员对手稿的批判性阅读。这项工作得到了创新领域科学研究补助金 (#26113005 s. n. 和 jp17h05679 至 s. i.) 的部分支持;a 日本教育、科学、体育和文化部为青年科学家提供补助金 (a) (jp17h04998 至 s. i.);和创业项目 “细胞进化” 和所有 riken 项目 “疾病和表观体” 从 riken (到 s. n. 和 s. i.)。
Materials
| Protein LoBind tube, 2 mL | Eppendorf | No. 0030108132 | For cell lysis |
| Protein LoBind tube, 1.5 mL | Eppendorf | No. 0030108116 | For ChIP and library preparation |
| DNA LoBind tube, 1.5 mL | Eppendorf | No. 0030108051 | For ChIP and library preparation |
| 8-strip PCR tube | BIO-BIK | 3247-00 | For ChIP and library preparation |
| SK Mill | TOKKEN | SK-200 | Handy cryogenic grinder to make cell powder for fixation |
| Metal bullet | TOKKEN | SK-100-DLC10 | Accessory of SK Mill |
| 2 mL stainless steel tube | TOKKEN | TK-AM5-SUS | An option for cell lysis |
| 2 mL stainless steel tube holder | TOKKEN | SK-100-TL | An option for cell lysis |
| 16% formaldehyde (w/v), methanol-free | Pierce | 28906 | To fix cells. Prepare 1% solution before use. |
| Glycine | Nacalai Tesque | 17109-35 | Prepare 2.5 M stock |
| D-PBS (-)(1x) | Nacalai Tesque | 14249-24 | For washing lysate and purified DNA |
| HEPES | Nacalai Tesque | 02443-05 | For Lysis buffer 1. Prepare 1 M, pH 7.5 stock. |
| 5 M NaCl, molecular biology grade | Nacalai Tesque | 06900-14 | For Lysis buffer 1, Lysis buffer 2, ChIP Elution Buffer, and Tris-EDTA-NaCl Buffer |
| 0.5 M EDTA, molecular biology grade | Wako Pure Chemical Industries, Ltd. | 311-90075 | For Lysis buffer 1, Lysis buffer 2, ChIP Elution Buffer, and Tris-EDTA-NaCl Buffer |
| Glycerol | Wako Pure Chemical Industries, Ltd. | 072-04945 | For lysis buffer 1 |
| NP-40 | Nacalai Tesque | 25223-75 | For lysis buffer 1 |
| Triton X-100, molecular biology grade | Nacalai Tesque | 12967-32 | For Lysis buffer 1 |
| Tris | Nacalai Tesque | 35406-91 | For Lysis buffer 2, ChIP Elution Buffer, and Tris-EDTA-NaCl Buffer. Prepare 1 M, pH 8.0 stock. |
| 0.1 M EGTA pH neutral | Nacalai Tesque | 08947-35 | For Lysis Buffer 2 |
| Protease inhibitor cocktail (100x) | Nacalai Tesque | 25955-24 | To block degradation of protein |
| RIPA buffer | Thermo Fisher Scientific | 89900 | For cell lysis and washing |
| milliTUBE 1 mL AFA Fiber | Covaris | 520130 | Sonicator tube. Accessory of Focused-ultrasonicator |
| Focused-ultrasonicator | Covaris | S220 or E220 | To digest DNA into adequate size for ChIP-Seq |
| UltraPure 10% SDS | Thermo Fisher Scientific | 15553-027 | For ChIP Elution Buffer |
| RNase A | Nacalai Tesque | 30141-14 | To purify DNA from lysate |
| Proteinase K, recombinant, PCR Grade | Sigma-Aldrich | 3115887001 | To purify DNA from lysate |
| Ethanol | Wako Pure Chemical Industries, Ltd. | 054-07225 | Make 70% solution |
| Monoclonal anti-FLAG M2 antibody produced in mouse | Sigma-Aldrich | F1804 | To purify chromatin expressed in cells of interest |
| Dynabead M-280 Sheep Anti-Mouse IgG | Thermo Fisher Scientific | 11201D | This can be used for anti-FLAG IP and anti-H3K4me3 IP |
| Anti-tri-methyl histone H3 (K4), mouse monoclonal antibody | Wako Pure Chemical Industries, Ltd. | 301-34811 | Any other antibody that works for ChIP analysis will work |
| 10x Blocking Reagent | Sigma-Aldrich | 11096176001 | For blocking during affinity purification |
| Denhardt’s solution | Nacalai Tesque | 10727-74 | For blocking during affinity purification |
| Glycogen (5 mg/ml) | Thermo Fisher Scientific | AM9510 | To purify DNA from lysate |
| Qubit 2.0 Fluorometer | Thermo Fisher Scientific | Q32866 | For quantification of isolated DNA |
| Qubit dsDNA HS Assay Kit | Thermo Fisher Scientific | Q32851 | For quantification of isolated DNA |
| 0.5 mL tube | Axygen | 10011-830 | For quantification by Qubit |
| Phenol/chloroform/isoamyl alcohol (25:24:1) | Nacalai Tesque | 25970-56 | To purify DNA from lysate |
| AMPure XP beads | Beckman Coulter | A63881 | SPRI magnetic beads for library preparation |
| Metal ice rack | Funakoshi | IR-1 | To keep the cell lysate frozen |
| Sample Cooler | New England Biolabs | T7771S | Helps fix cells with minimal damage |
| 2100 Bioanalyzer | Agilent Technologies | G2939BA | To check the quality of isolated DNA fragments. Another fragment analyzer can be used. |
| Bioanalyzer 2100 Expert Software | Agilent Technologies | G2946CA | Supplied with the Bioanalyzer |
| High Sensitivity DNA Kit | Agilent Technologies | 5067-4626 | To check the quality of the isolated DNA fragments |
| KAPA LTP Library Preparation Kit | Roche | 07961898001 | Supplied with 10x KAPA End Repair Buffer, KAPA End Repair Enzyme Mix, KAPA A-Tailing Buffer, KAPA A-Tailing Enzyme, KAPA Ligation Buffer, KAPA DNA Ligase, and PEG/NaCl solution |
| NEXTflex DNA Barcodes | BIOO Scientific | NOVA-514101 | Adapter for library preparation. Supplied with DNA Barcode Adapters and Primer Mix. |
| KAPA Real-Time Library Amplification Kit | Roche | 07959028001 | Supplied with 2x KAPA HiFi HS real-time PCR Master Mix, PCR Primer Mix, and Fluorescent Standards |
| 2x KAPA HiFi HotStart ReadyMix | Roche | KM2602 | For library preparation. Additionally, this enzyme may be required for the KAPA Real-Time Library Amplification Kit |
| Buffer EB | Qiagen | 19086 | 10 mM Tris-Cl, pH 8.5 for elution of DNA |
| 386-well qPCR plate | Thermo Fisher Scientific | 4309849 | For real-time PCR |
| QuantStudio 7 Flex Real-Time PCR System | Thermo Fisher Scientific | 4485701 | To quantify DNA |
| MicroAmp Optical Adhesive Film | Thermo Fisher Scientific | 4311971 | For real-time PCR |
| MicroAmp Clear Adhesive Film | Thermo Fisher Scientific | 4306311 | For plate sealing |
| End-repair master mix | Combine 1.4 µL of 10x KAPA End Repair Buffer, 1 µL of KAPA End Repair Enzyme Mix, and 1.6 µL of H2O | ||
| A-taling master mix | Combine 1 µL of KAPA A-Tailing Buffer, 0.6 µL of KAPA A-Tailing Enzyme, and 8.4 µL of H2O | ||
| Ligation buffer mix | Combine 2 µL of KAPA ligation buffer and 6 µL of H2O | ||
| Real-time PCR master mix | Combine 5 µL of 2x KAPA HiFi HS real-time PCR Master Mix, 0.35 µL of PCR Primer Mix (10 µM each of forward primer AATGATACGGCGACCACCGAG and reverse primer CAAGCAGAAGACGGCATACGAG), and 3.15 µL of H2O | ||
| PCR master mix | Combine 10 µL of 2x KAPA HiFi Ready Mix, 0.9 µL of PCR Primer Mix, and 0.6 µL of H2O | ||
| Integrative Genomics Viewer | Broad Institute | IGV_2.3.88 | Genome browser to visualize sequencing data |
| DNA olgionucleotide: 5′-GCCTACGCAGGTCTTGCTGAC-3′ | Eurofins Genomics | A primer to amplify the promoter region of GAPDH | |
| DNA olgionucleotide: 5′-CGAGCGCTGACCTTGAGGTC-3′ | Eurofins Genomics | A primer to amplify the promoter region of GAPDH | |
| SYBR Premix Ex Taq | Takara | RR420L | To quantify the DNA corresponding to the GADPH promoter region |
| Thermal Cycler Dice | Takara | TP870 | To quantify the DNA corresponding to the GADPH promoter region |
Referencias
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Citar este artículo
Mito, M., Kadota, M., Nakagawa, S., Iwasaki, S. TChIP-Seq: Cell-Type-Specific Epigenome Profiling. J. Vis. Exp. (143), e58298, doi:10.3791/58298 (2019).