摘要:Hi-C技术源于染色体构象捕获 (Chromosome Conformation Capture, 3C) 技术,用于分析染色质三维空间结构的一种测序方法。将三维基因组用甲醛交联固定,用内切酶进行酶切后在DNA末端加生物素标记,使DNA与蛋白、蛋白与蛋白分开,然后,提取DNA并打断成小片段,用磁珠捕获带生物素标记的片段进行建库测序,对测序数据进行分析即可揭示染色体的远程相互作用,阐述染色体的三维构象和可能的基因之间的调控关系。该技术广泛用于分化发育机制、突变机制、疾病的发生发展等研究,也是近年来解析染色体水平的参考基因组的一个关键环节。
以枯叶蛱蝶属蝴蝶为例,利用Hi-C技术获得蝴蝶特定发育时期的基因组序列,结合生物信息分析方法,研究全基因组范围内整个染色质DNA在空间位置上的关系,从而获得高分辨率的染色质三维结构信息,为组装蝴蝶的染色体水平的参考基因组提供重要的数据。
关键词: 蝴蝶, Hi-C, 染色质, 三维结构
研究背景
Hi-C (High-through chromosome conformation capture) 技术 (Dekker et al., 2002) 源于染色体构象捕获 (conformation capture, 3C) 技术,以整个细胞核为研究对象,利用高通量测序技术,结合生物信息分析方法,研究全基因组范围内整个染色质DNA在空间位置上的关系,通过对染色质内部全部DNA相互作用模式进行捕获,获得高分辨率的染色质三维结构信息。2009年,Job Dekker研究团队 (Lieberman-Aiden et al., 2009) 首次提出了Hi-C测序的概念,并利用该技术得到人类正常淋巴细胞基因组的三维结构图。通过Hi-C技术可以获得全基因组范围内的互作信息,得到染色体三个层级的三维结构,即常染色质/异染色质 (A/B compartment)、拓扑相关结构域 (TAD)、染色质环 (loop);通过Hi-C技术可建立基因组折叠模型,研究染色体之间相互作用;Hi-C技术可应用于基因组组装、单体型构建等,并可以与RNA-seq、ChIP-seq等数据进行联合分析,从基因调控网络和表观遗传网络来阐述生物体性状形成的相关机制。Hi-C技术在蝴蝶中的应用也越来越广泛,例如,中国科学院昆明动物研究所进化基因组学与基因起源研究团队于2019年利用Hi-C技术结合三代长读长测序技术 (Pacbio) 完成了染色体水平的碧凤蝶 (Papilio bianor Cramer, 1777) 基因组组装 (29条常染色体和1条性染色体),这是首个利用Hi-C技术完成的染色体水平的蝴蝶基因组 (Lu et al., 2019);并于2020年利用Hi-C技术结合三代长读长测序技术 (Pacbio) 和核型实验成功解析了枯叶蛱蝶 (Kallima inachus Doyère, 1840) 染色体水平的高质量基因组 (30条常染色体及Z和W两条性染色体),这是首个组装了W染色体的蝴蝶参考基因组,也是至今组装得最完整的蝴蝶参考基因组 (Yang et al., 2020)。
本文参考Belton等 (Belton et al., 2012; Ma et al., 2015; Nagano et al., 2015; van Berkum et al., 2010) 的方法对非模式动物蝴蝶幼虫进行了Hi-C文库的构建。
材料与试剂
- 移液吸头
- 离心管
- 一次性培养皿
- 解剖刀
- 解剖剪
- 镊子
- 血球计数板
- Corning cell strainer 40 µm Nylon (BD)
- DynaMagTM-2磁力架 (Invitrogen, catalog number: 12321D)
- MilliQ ddH2O
- Tris饱和酚 (Solarbio, catalog number: T0250-250)
- 氯仿 (Mecklin, catalog number: C2432-500ML)
- 异戊醇 (Aladdin, catalog number: M116198-500ml)
- 琼脂糖 (Biowest, catalog number: 111860)
- EDTA (Solarbio, catalog number: E8040-500g)
- 1 M Tris-HCl (pH 8.0) (Coolaber, catalog number: SL3080-100mL)
- 3 M Sodium acetate (pH 5.2) (Thermo, catalog number: R1181)
- 2.5 M Glycine (Biofroxx, catalog number: 1275)
- NaCl (Solarbio, catalog number: S8210-500g)
- KCl (生工, catalog number: 231-211-8)
- Na2HPO4·12H2O (Sigma-Aldrich,catalog number: 1065790500)
- KH2PO4 (Macklin, catalog number: P815662-500g)
- NaHCO3 (Sigma-Aldrich, catalog number: S8875-500G)
- 酚红 (Macklin, catalog number: P6066-100g)
- 0.2% Igepal CA-630 (Sigma-Aldrich, catalog number: I8896-100ML)
- 100% Ethanol (Merck, catalog number: 459844-2.5 L)
- TE Buffer (pH 8.0) (Solarbio, catalog number: T1120)
- BSA (Solarbio, catalog number: A8010-5g)
- 10% (w/v) Triton X-100 (Tiandz, catalog number: 25-07910)
- 10% (w/v) SDS (Simgen, catalog number: 9021100)
- RNase A (康为世纪, catalog number: CW0601S)
- Tween-20 (Coolaber, catalog number: CT11551-100ml)
- 0.25% Trypsin-EDTA (Gibco, catalog number: 25200-072)
- Calf serum (ExCell, catalog number: NCD100)
- DMEM (BI, catalog number: 14417)
- 1× Phosphate-buffered saline (PBS) (pH 7.2) (Cell Signaling, catalog number: 9872L)
- 37% Formaldehyde (Sigma Aldrich, catalog number: 1040031000)
- Proteinase K (10 mg/ml) (Coolaber, catalog number: SL2074-1mL)
- HaltTM Protease Inhibitor Cocltail (100×) (Thermo Scientific, catalog number: 87786)
- Klenow DNA Polymerase I (NEB, catalog number: M0210S)
- Klenow Fragment (3’ → 5’ exo-) (NEB, catalog number: M0212S)
- T4 DNA Ligase (NEB, catalog number: M0202V)
- T4 DNA Polymerase (NEB, catalog number: M0203L)
- T4 Polynucleotide Kinase (NEB, catalog number: M0201V)
- Dpn II (NEB, catalog number: R0543S)
- NEBNext Multiplex Oligos for Illumina (NEB, catalog number: E7535S)
- 10× NEBuffer 3.1 (NEB, catalog number: B7203S)
- 5× Ligation Buffer (Invitrogen, catalog number: 46300-018)
- 10× NEBuffer 2.1 (NEB, catalog number: B7202V)
- 10× Ligation Buffer (NEB, catalog number: B0202S)
- User Enzyme (NEB, catalog number: M5505S)
- Agencourt AMPure XP Beads (Beckman Coulter, catalog number: A63881)
- VAHTS Adapter for Illumina (Vazyme, catalog number: ND104-01-AM)
- VATHS Universal PCR Primer for Illumina (Vazyme, catalog number: ND104-01-AN)
- VATHS Index Primer for Illumina (Vazyme, catalog number: ND104-01)
- Dynabeads MyOne™ Streptavidin C1 beads (Invitrogen, catalog number: 65001)
- 25 mM dNTP mix (TaKaRa, catalog number: SD0304)
- 10 mM dATP,10 mM dGTP,10 mM dCTP,10 mM dTTP (Invitrogen, catalog number: 18252015, 18254011, 18253013, 18255018)
- 0.4 mM biotin-14-dATP (Invitrogen, catalog number: 19524-016)
- Qubit dsDNA HS Assay Kit (Invitrogen, catalog number: Q32851)
- Qubit dsDNA BR Assay Kit (Invitrogen, catalog number: Q32850)
- Agilent High Sensitivity DNA Kit (Agilent, catalog number: 5067-4626)
- Phusion DNA Polymerase (Stratagene, catalog number: 600670-51)
- 10× PfuUltra Buffer (Stratagene, 600670-52)
- D-Hanks缓冲液
- 1× Lysis缓冲液
--- Stored at 4 °C
- Tween Wash Buffer (TWB)
- 2× Binding Buffer (BB)
仪器设备
- 恒温水浴摇床 (GFL, model: 1092)
- 冷冻离心机 (Eppendorf Centrifuge, model: 5810 R)
- 体视显微镜 (Nikon, model: SMZ745)
- 超声波DNA破碎仪 (Covaris, model: M220)
- ThermoMixerF 1.5精巧型恒温混匀仪 (Eppendorf, model: 5384000071)
- ProFlex PCR System (Thermofisher, model: 4484075)
- QubitTM 4 荧光计 (Invitrogen, catalog number: Q33238)
- Agilent 2100 Bioanalyzer System (Agilent, model: G2939BA)
实验步骤
- 分离细胞 (Cell Separation)注:不同物种分离细胞所获得的细胞量不同,在正式实验之前应进行分离细胞预实验,以便评估可获得的细胞数量。若细胞量远大于1×108时,则加入2 ml 1×
PBS,减少因细胞量过多而引起的细胞计数误差;若细胞量低于1×108时,则加入1 ml左右的1× PBS,以便更好的进行后续实验。 - 细胞交联 (Crosslinking of Cells)以上即为交联后样品,可用于接下来的实验,或者-80 °C冻存,冻存样品3个月内使用。
- 细胞裂解和染色体消化 (Cell Lysis and Chromatin Digestion)
- 末端加生物素标记 (Marking of DNA Ends with Biotin-14-dCTP)
- 平端连接 (Blunt End Ligation)
- 反交联 (Reverse Crosslinking)
- DNA纯化 (DNA Purification)注: 所获得的DNA样品可于-20°C保存。
- 去除末端未连接生物素 (Removal of Biotin from Un-ligated Ends)
- DNA打断 (DNA Sonication)
- DNA片段筛选 (Size Fractionation)(可选做) 取样品使用2%琼脂糖凝胶检测打断及片段选择的片段大小范围。
- 磁珠捕获携带生物素片段 (Biotin Pulldown with Streptavidin Coated Beads)
- 末端补平 (End Repair)
- 末端加 “A” (A-Tailing)
- 接头连接 (Adaptor Ligation)
- User酶处理 (User Enzyme)
- PCR扩增 (PCR Amplification)
- PCR产物纯化及片段筛选 (Production PCR and Size Selection)
- Hi-C文库质控 (Quality Control of Hi-C Library)
- Hi-C文库测序 (Hi-C Sequencing)
所获得的Hi-C文库使用Illumina HiseqXten (PE150) 平台进行上机测序,测序深度根据物种的基因组大小和项目的设计进行确定。在蝴蝶基因组研究中数据量通常按物种基因组大小的100× 进行测序。
致谢
感谢国家自然科学基金委员会创新研究群体 (31621062)、国家自然科学基金委员会面上项目 (32070482)、中国科学院西部之光项目 (A类) 对中国科学院昆明动物研究所遗传资源与进化国家重点实验室的支持。
参考文献
- Belton, J. M., McCord, R. P., Gibcus, J. H., Naumova, N., Zhan, Y. and Dekker, J. (2012). Hi-C: a comprehensive technique to capture the conformation of genomes. Methods 58 (3): 268–276.
- Dekker, J., Rippe, K., Dekker, M. and Kleckner, N. (2002). Capturing chromosome conformation. Science 295 (5558): 1306–1311.
- Lieberman-Aiden, E., van Berkum, N.L., Williams, L., Imakaev, M., Ragoczy, T., Telling, A., Amit, I., et al. (2009). Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326: 289–293.
- Lu, S., Yang, J., Dai, X., Xie, F., He, J., Dong, Z., Mao, J., Liu, G., Chang, Z., Zhao, R., Wan, W., Zhang, R., Li, J., Wang, W. and Li, X. (2019). Chromosomal-level reference genome of Chinese peacock butterfly (Papilio bianor) based on third-generation DNA sequencing and Hi-C analysis. GigaScience 8: 128.
- Ma, W., Ay, F., Lee, C., Gulsoy, G., Deng, X., Cook, S., Hesson, J., Cavanaugh, C., Ware, C.B., Krumm, A., Shendure, J., Blau, C. A., Disteche, C. M., Noble, W. S. and Duan, Z. (2015). Fine-scale chromatin interaction maps reveal the cis-regulatory landscape of human lincRNA genes. Nat Methods 12: 71–78.
- Nagano, T., Lubling, Y., Yaffe, E., Wingett, S.W., Dean, W., Tanay, A. and Fraser, P. (2015). Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell. Nat Protoc 10: 1986–2003.
- van Berkum, N.L., Lieberman-Aiden, E., Williams, L., Imakaev, M., Gnirke, A., Mirny, L.A., Dekker, J. and Lander, E.S. (2010). Hi-C: a method to study the three-dimensional architecture of genomes. J Vis Exp (39): 1869.
- Yang, J., Wan, W., Xie, M., Mao, J., Dong, Z., Lu, S., He, J., Xie, F., Liu, G., Dai, X., Chang, Z., Zhao, R., Zhang, R., Wang, S., Zhang, Y., Zhang, W., Wang, W. and Li, X. (2020). Chromosome-level reference genome assembly and gene editing of the dead-leaf butterfly Kallima inachus. Mol Ecol Resour 20(4): 1080–1092.
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:万雯婷, 常洲, 董志巍, 王文, 李学燕. (2021). 蝴蝶Hi-C文库构建.
Bio-101: e1010654. DOI:
10.21769/BioProtoc.1010654.
How to cite: Wan, W.T., Chang, Z., Dong, Z.W., Wang, W. and Li, X.Y. (2021). Construction of Hi-C Library for Butterfly.
Bio-101: e1010654. DOI:
10.21769/BioProtoc.1010654.