CARIP 和芯片序列: 鉴定胚胎干细胞中染色质相关 rna 和蛋白质-DNA 相互作用的方法
Summary
在这里, 我们描述了执行芯片序列和 CARIP 的方法, 包括用于下一代测序的库准备, 以生成 ES 细胞中的全球 epigenomic 和染色质相关的 RNA 图谱。
Abstract
胚胎干细胞的自我更新和分化受外部信号和转录因子的内在网络、表观遗传调控、combinatorially 对基因组蛋白的转换后修饰的制约。附近基因的表达状态。RNA 也被证明与各种各样的蛋白质交互调控染色质动力学和基因表达。染色质相关 RNA 免疫沉淀 (CARIP) 其次是下一代测序 (CARIP 序列) 是一种新的方法来调查与染色质蛋白相关的 rna, 而染色质免疫沉淀其次是下一代测序 (芯片序列) 是一种功能强大的基因组学技术, 用于在 ES 细胞的全球尺度上绘制出蛋白、转录因子和表观遗传修饰符的平移后修饰位置。在这里, 我们描述了执行 CARIP 和芯片序列的方法, 包括用于下一代测序的库构造, 以生成 ES 细胞中的全球染色质相关 RNA 和 epigenomic 图。
Introduction
胚胎干细胞的命运决定由细胞外信号和一系列转录调控器之间的通讯调节, 其中包括组蛋白修饰语和组蛋白尾的翻译后修饰。这些相互作用促进染色质可达性和染色质的包装成两种状态之一: 染色质, 是开放和转录活跃的, 染色质, 这是紧凑的, 一般转录不活跃。转录因子与 DNA 序列特异的结合亲和性和表观遗传修饰, euchromatic 区域参与控制基因表达。下一代测序方法, 包括芯片序列1, 在映射全基因组转录网络 (ES 细胞自我更新和干细胞2、3、4) 方面发挥了重要作用. ,5,6。此外, 虽然 rna immunopreciation 其次是下一代测序 (RIP 序列)7对 RNA-蛋白质相互作用的评价表明, DNA 结合蛋白与 rna 相互作用来调节转录事件7,8,9,10,11,12, 很少有研究调查了与染色质12相关的 rna 的全基因组定位, 或者 rna 和组蛋白修饰之间的全球相互作用。长非编码 rna (lncRNAs) 是一种 rna, 它已被发现来调节染色质相关蛋白的活性13,14,15。例如, Xist 是一种 lncRNA, 通过招募阻遏16、17, 在雌性哺乳动物细胞中调节一个 X 染色体的失活。然而, 与染色质相关的 rna 的全谱在很大程度上是未知的。在这里, 我们描述了一个新的协议, 染色质相关的 RNA 免疫沉淀 (CARIP) 其次是下一代测序 (CARIP 序列), 以确定染色质相关 rna 在全基因组的基础上的 ES 细胞, 包括图书馆准备下一代测序, 和芯片序列, 以映射全球占用的组蛋白修饰, 转录因子, 和表观遗传修饰。与其他 RIP 序列方法7不同, CARIP 序列包括交联和超声波步骤, 它允许直接识别与染色质相关的 rna。在一起, 芯片序列是一个强有力的工具, 以确定全基因组范围内的蛋白质-DNA 相互作用, 而 CARIP 是一个强大的方法来调查与染色质成分相关的 rna。
Protocol
1. 小鼠 ES 细胞在无饲养条件下的培养。 注意: 小鼠 ES 细胞是传统的在培养基上培养的细胞培养皿上涂有明胶和单层小鼠胚胎成纤维细胞 (MEF), 已 mitotically 灭活 (iMEFs)。然而, MEFs 应在下游表观或表达分析之前被删除, 以防止与 MEF 相关的染色质和 RNA 的污染。 给料层的制备: 在水中加入2毫升0.1% 的明胶, 并在37摄氏度下孵育20-30 分钟, 明胶涂层 a 6 井细胞培养板。 ?…
Representative Results
我们通过这个芯片序列协议6成功地审问了 ES 细胞中 H3K4me3、H3K4me2 和 KDM5B 的全基因组绑定。ES 细胞是在无馈线条件下培养的 (图 1), 并如上文所述交叉链接。超声波随后按照芯片序列协议步骤3.2 所述执行, 并通过在2% 琼脂糖凝胶上运行 DNA 进行评估 (图 2)。接下来, 按照步骤4中的描述执行芯片, 并按照步骤6中的?…
Discussion
在 ES 细胞中, 芯片序列是评价全球蛋白质-DNA 相互作用 (e. g)、转录因子/组蛋白修饰酶/组蛋白修饰和 DNA 的有效方法, 而新开发的 CARIP 序列协议在审问基因组范围内的 rna 与染色质成分的关联。芯片序列是一种基本的工具, 用于评估 ES 细胞和其他细胞类型的表观景观。芯片序列和 CARIP 库的质量在很大程度上依赖于芯片级抗体。对芯片序列的抗体的适用性可以通过执行芯片 PCR 的经验来确定, ?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了韦恩州立大学 Karmanos 癌症研究所的支持, 由国家心脏、肺和血液研究所 (1K22HL126842-01A1) 授予 B.L.K。这项工作利用韦恩州立大学高性能计算网格计算资源 ()。我们感谢集集 Kurup 对 CARIP 数据分析的帮助。
作者的贡献:
B.L.K. 构思了 CARIP 法, 设计并进行了芯片序列和 CARIP 的实验, 分析了测序数据, 起草了手稿。所有作者都已阅读并批准了本手稿的最终版本。
Materials
| Mouse leukemia inhibitory factor (ESGRO LIF) | EMD Millipore | 106 or 107 units | |
| GSK3β inhibitor CHIR99021 (GSK3i) | Stemgent | Solvent: DMSO 10mM | |
| MEK inhibitor PD0325901 (MEKi) | Stemgent | Solvent: DMSO 10mM | |
| Dulbecco's Modified Eagle Medium (DMEM), high glucose | Invitrogen | 11965092 | |
| PBS (phosphate buffered saline) | Invitrogen | 10010031 | |
| Trypsin-EDTA (0.25%), phenol red | Gibco | 25200056 | |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140122 | |
| 2-Mercaptoethanol (50 mM) | Gibco | 31350010 | |
| MEM Non-Essential Amino Acids Solution (100X) | Gibco | 11140076 | |
| EmbryoMax ES Cell Qualified Fetal Bovine Serum, 500 ml | EMD Millipore | ES-009-B | |
| EmbryoMax 0.1% Gelatin Solution | EMD Millipore | ES-006-B | |
| Glycine | Sigma | G7126-500G | 1.25 M stock conentration in water |
| Formaldehyde solution | Sigma | F8775-500ML | |
| TE (Tris EDTA) pH 8.0 1X | Quality Biological | 351-011-131 | |
| Phenylmethanesulfonyl fluoride (PMSF) solution | Sigma | 93482-250ML-F | |
| cOmplete Protease Inhibitor Cocktail | Roche | 11697498001 | |
| Sodium dodecyl sulfate solution (20%) | Quality Biological | A611-0837-10 | |
| Q125 sonifier | Qsonica | 4422 | |
| Triton X-100 solution | Sigma | 93443-100ML | |
| Sodium deoxycholate | Sigma | 30970 | |
| NaCl | Sigma | S7653 | |
| Dynabeads Protein G for Immunoprecipitation | Invitrogen | 10004D | |
| Dynabeads Protein A for Immunoprecipitation | Invitrogen | 10002D | |
| Dynabeads Protein A/Protein G and Magnet Starter Pack | Invitrogen | 10015D | |
| Lithium chloride | Sigma | L4408 | |
| IGEPAL CA-630 | Sigma | I8896 | |
| RNeasy Mini Kit | Qiagen | 74104 | |
| Dynabeads mRNA Purification Kit (for mRNA purification from total RNA preps) | Invitrogen | 61006 | |
| SuperScript Double-Stranded cDNA Synthesis Kit | Invitrogen | 11917010 | |
| QIAquick PCR Purification Kit | Qiagen | 28104 | |
| End-It DNA End-Repair Kit | Lucigen | ER81050 | |
| Klenow Fragment (3'→5' exo-) | NEB | M0212L | |
| dATP (10 mM) | Invitrogen | 18252015 | |
| T4 DNA ligase | NEB | M0202L | |
| TrackIt 1 Kb Plus DNA Ladder | Invitrogen | 10488085 | |
| E-gel EX agarose gels, 2% | Invitrogen | G402002 | |
| Phusion high-fidelity 2X master mix with HF buffer | Thermo Fisher | F531LPM | |
| ChIPAb+ Trimethyl-Histone H3 (Lys4) – ChIP Validated Antibody | EMD Millipore | 17-614 | |
| Anti-Histone H3 (di methyl K4) antibody [Y47] – ChIP Grade (ab32356) | Abcam | ab32356 | |
| Corning Costar Flat Bottom Cell Culture Plates (6-well) | Fisher | 720083 | |
| Falcon Standard Tissue Culture Dishes (10cm) | Fisher | 08772E | |
| Bioruptor pico | Diagenode | B01010001 | |
| Qubit Flurometer 2.0 | Thermo Fisher | ||
| Qubit dsDNA HS Assay Kit | Thermo Fisher | Q32851 | |
| MinElute Reaction Cleanup Kit | Qiagen | 28204 | |
| MinElute Gel Extraction Kit | Qiagen | 28604 | |
| Anti-Histone H4 (tri methyl K20) antibody – ChIP Grade (ab9053) | Abcam | ab9053 | |
| Labquake rotator | Thermo Fisher | 400110Q | |
| Illumina HiSeq platform | Illumina | ||
| TURBO DNase | Invitrogen | AM2238 |
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Tags
Keywords:
CARIP-SeqChIP-SeqChromatin-associated RNAsProtein-DNA InteractionsEmbryonic Stem CellsEpigeneticsTranscription FactorsEpigenetic RegulatorsHistone ModificationsChromatin ImmunoprecipitationSonicationFormaldehyde Cross-linkingGlycinePhosphate Buffered SalineTrypsinizationMEF MediumFetal Bovine SerumSonication BufferSodium Dodecyl Sulfate
Citazione di questo articolo
Kidder, B. L. CARIP-Seq and ChIP-Seq: Methods to Identify Chromatin-Associated RNAs and Protein-DNA Interactions in Embryonic Stem Cells. J. Vis. Exp. (135), e57481, doi:10.3791/57481 (2018).