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。
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。
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。
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?…
The authors have nothing to disclose.
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.)。
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 |