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

1. 移液吸头
2. 离心管
3. 一次性培养皿
4. 解剖刀
5. 解剖剪
6. 镊子
7. 血球计数板
8. Corning cell strainer 40 µm Nylon (BD)
9. DynaMagTM-2磁力架 (Invitrogen, catalog number: 12321D)
10. MilliQ ddH₂O
11. Tris饱和酚 (Solarbio, catalog number: T0250-250)
12. 氯仿 (Mecklin, catalog number: C2432-500ML)
13. 异戊醇 (Aladdin, catalog number: M116198-500ml)
14. 琼脂糖 (Biowest, catalog number: 111860)
15. EDTA (Solarbio, catalog number: E8040-500g)
16. 1 M Tris-HCl (pH 8.0) (Coolaber, catalog number: SL3080-100mL)
17. 3 M Sodium acetate (pH 5.2) (Thermo, catalog number: R1181)
18. 2.5 M Glycine (Biofroxx, catalog number: 1275)
19. NaCl (Solarbio, catalog number: S8210-500g)
20. KCl (生工, catalog number: 231-211-8)
21. Na₂HPO₄·12H₂O (Sigma-Aldrich，catalog number: 1065790500)
22. KH₂PO₄ (Macklin, catalog number: P815662-500g)
23. NaHCO₃ (Sigma-Aldrich, catalog number: S8875-500G)
24. 酚红 (Macklin, catalog number: P6066-100g)
25. 0.2% Igepal CA-630 (Sigma-Aldrich, catalog number: I8896-100ML)
26. 100% Ethanol (Merck, catalog number: 459844-2.5 L)
27. TE Buffer (pH 8.0) (Solarbio, catalog number: T1120)
28. BSA (Solarbio, catalog number: A8010-5g)
29. 10% (w/v) Triton X-100 (Tiandz, catalog number: 25-07910)
30. 10% (w/v) SDS (Simgen, catalog number: 9021100)
31. RNase A (康为世纪, catalog number: CW0601S)
32. Tween-20 (Coolaber, catalog number: CT11551-100ml)
33. 0.25% Trypsin-EDTA (Gibco, catalog number: 25200-072)
34. Calf serum (ExCell, catalog number: NCD100)
35. DMEM (BI, catalog number: 14417)
36. 1× Phosphate-buffered saline (PBS) (pH 7.2) (Cell Signaling, catalog number: 9872L)
37. 37% Formaldehyde (Sigma Aldrich, catalog number: 1040031000)
38. Proteinase K (10 mg/ml) (Coolaber, catalog number: SL2074-1mL)
39. Halt^TM Protease Inhibitor Cocltail (100×) (Thermo Scientific, catalog number: 87786)
40. Klenow DNA Polymerase I (NEB, catalog number: M0210S)
41. Klenow Fragment (3’ → 5’ exo-) (NEB, catalog number: M0212S)
42. T4 DNA Ligase (NEB, catalog number: M0202V)
43. T4 DNA Polymerase (NEB, catalog number: M0203L)
44. T4 Polynucleotide Kinase (NEB, catalog number: M0201V)
45. Dpn II (NEB, catalog number: R0543S)
46. NEBNext Multiplex Oligos for Illumina (NEB, catalog number: E7535S)
47. 10× NEBuffer 3.1 (NEB, catalog number: B7203S)
    
48. 5× Ligation Buffer (Invitrogen, catalog number: 46300-018)
49. 10× NEBuffer 2.1 (NEB, catalog number: B7202V)
50. 10× Ligation Buffer (NEB, catalog number: B0202S)
51. User Enzyme (NEB, catalog number: M5505S)
52. Agencourt AMPure XP Beads (Beckman Coulter, catalog number: A63881)
53. VAHTS Adapter for Illumina (Vazyme, catalog number: ND104-01-AM)
54. VATHS Universal PCR Primer for Illumina (Vazyme, catalog number: ND104-01-AN)
55. VATHS Index Primer for Illumina (Vazyme, catalog number: ND104-01)
56. Dynabeads MyOne™ Streptavidin C1 beads (Invitrogen, catalog number: 65001)
57. 25 mM dNTP mix (TaKaRa, catalog number: SD0304)
58. 10 mM dATP，10 mM dGTP，10 mM dCTP，10 mM dTTP (Invitrogen, catalog number: 18252015, 18254011, 18253013, 18255018)
59. 0.4 mM biotin-14-dATP (Invitrogen, catalog number: 19524-016)
60. Qubit dsDNA HS Assay Kit (Invitrogen, catalog number: Q32851)
61. Qubit dsDNA BR Assay Kit (Invitrogen, catalog number: Q32850)
62. Agilent High Sensitivity DNA Kit (Agilent, catalog number: 5067-4626)
63. Phusion DNA Polymerase (Stratagene, catalog number: 600670-51)
64. 10× PfuUltra Buffer (Stratagene, 600670-52)
65. D-Hanks缓冲液
    
    
    
    
66. 1× Lysis缓冲液
    
    
    
    --- Stored at 4 °C
    
67. Tween Wash Buffer (TWB)
    
    
    
    
68. 2× Binding Buffer (BB)
    
    
    
    

仪器设备

1. 恒温水浴摇床 (GFL, model: 1092)
2. 冷冻离心机 (Eppendorf Centrifuge, model: 5810 R)
3. 体视显微镜 (Nikon, model: SMZ745)
4. 超声波DNA破碎仪 (Covaris, model: M220)
5. ThermoMixerF 1.5精巧型恒温混匀仪 (Eppendorf, model: 5384000071)
6. ProFlex PCR System (Thermofisher, model: 4484075)
7. Qubit^TM 4 荧光计 (Invitrogen, catalog number: Q33238)
8. Agilent 2100 Bioanalyzer System (Agilent, model: G2939BA)
   

实验步骤