用于迭代表观基因组分析的可重复使用的单细胞
1Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute,National Institutes of Health, 2Biostatistics and Data Management Section, Center for Cancer Research, National Cancer Institute,National Institutes of Health, 3CCR Collaborative Bioinformatics Resource (CCBR), Center for Cancer Research, National Cancer Institute,National Institutes of Health, 4Advanced Biomedical Computational Science,Frederick National Laboratory for Cancer Research sponsored by the National Cancer Institute
Summary
本协议描述了一种使用可重复使用的单细胞进行迭代表观基因组分析的单细胞方法。可重复使用的单细胞允许分析同一单细胞中的多个表观遗传标记并对结果进行统计验证。
Abstract
目前的单细胞表观基因组分析是为一次性使用的而设计的。该细胞在单次使用后被丢弃，防止分析单个细胞中的多个表观遗传标记，并需要来自其他细胞的数据来区分单个细胞中的信号和实验背景噪声。本文描述了一种重复使用同一单细胞进行迭代表观基因组分析的方法。
在这种实验方法中，细胞蛋白首先锚定在聚丙烯酰胺聚合物上，而不是将它们与蛋白质和DNA交联，从而减轻结构偏差。这一关键步骤允许使用相同的单细胞重复实验。接下来，将具有用于邻近连接的支架序列的随机引物退火到基因组 DNA 上，并使用 DNA 聚合酶通过延伸将基因组序列添加到引物中。随后，针对表观遗传标记物和对照IgG的抗体（每个抗体用不同的DNA探针标记）与同一单细胞中的相应靶标结合。
通过在随机引物和抗体-DNA探针的支架序列中添加具有互补序列的连接器DNA，诱导随机引物和抗体之间的邻近连接。这种方法将抗体信息和附近的基因组序列整合到邻近连接的单个DNA产物中。通过对同一单细胞进行重复实验，该方法可以增加稀有细胞的数据密度，并仅使用来自同一细胞的IgG和抗体数据进行统计分析。通过该方法制备的可重复使用的单细胞可以存储至少几个月，并在以后重复使用以扩大表观遗传学表征并提高数据密度。这种方法为研究人员及其项目提供了灵活性。
Introduction
单细胞技术正在进入单细胞多组学时代，它集成了单个单细胞组学技术1。最近，单细胞转录组学已与检测染色质可及性（scNMT-seq2 和SHARE-seq3）或组蛋白修饰（Paired-seq4 和Paired-Tag5）的方法相结合。最近，单细胞转录组学和蛋白质组学与染色质可及性（DOGMA-seq6）相结合。这些方法使用基于转座酶的标记来检测染色质可及性或组蛋白修饰。
基于转座酶的方法切割基因组DNA，并在基因组DNA片段的末端添加DNA条形码。每个切割的基因组片段最多只能接受两个DNA条形码（=每个切割位点一个表观遗传标记），并且切割位点的基因组DNA丢失。因此，基于切割的方法需要在测试的表观遗传标记数量和信号密度之间进行权衡。这阻碍了对同一单细胞中多个表观遗传标记的分析。开发了一种不切割基因组DNA的单细胞表观基因组方法来克服这个问题7，8。
除了上面提到的切割衍生问题外，基于转座酶的方法还有其他局限性。在单细胞表观基因组分析中，了解组蛋白和DNA相关蛋白在基因组上的位置至关重要。在目前的方法中，这是通过使用未固定的单细胞和保留蛋白质 – DNA和蛋白质 – 蛋白质相互作用来实现的。然而，即使在组蛋白修饰的分析中，这也会对可接近的染色质区域产生强烈的偏差9。使用聚丙烯酰胺支架7，8，可以在不交联蛋白质-DNA和蛋白质-蛋白质的情况下保留组蛋白和基因组相关蛋白在基因组上的位置。这种方法减少了在当前依赖于蛋白质-DNA和蛋白质-蛋白质相互作用的方法中观察到的结构偏差。
基于转座酶的方法只能从单个细胞获取一次信号。因此，由于信号的下降，很难描绘出单个细胞的完整表观基因组。已经开发出可重复使用的单细胞，通过允许在同一单细胞中进行迭代表观基因组分析来克服当前的局限性。
Protocol
注意：该方法的示意图如图 1所示。 图 1：协议工作流程的示意图。 步骤7.2-13通过示意图进行说明。每行表示协议中的一个步骤。绿色的细胞蛋白是基于晶体结构（PDB：6M4G）产生的人核小体。 <a href="https://www.jove.com/files/ftp_upload/…
Representative Results
使用步骤8中描述的方案生成K562单细胞（见 图5）。单细胞嵌入聚丙烯酰胺珠的外层。使用插入器染料对细胞DNA进行染色和可视化。 图 5：生成的可重复使用的单个细胞。 用用于DNA的插层染料（绿色荧光）对细胞进行染色。白色箭?…
Discussion
本文介绍了最近报道的使用可重复使用的单细胞7进行单细胞多表观基因组分析的分步方案。在随后的段落中，我们将讨论关键点，强调协议中的潜在限制。
整个协议（从步骤7.2-13开始）的关键点之一是避免DNase污染。单个细胞只有两个基因组DNA拷贝。因此，破坏基因组DNA会严重减少信号数量。避免DNA酶污染对于保护基因组DNA的完整性至关重要。
<p class="…Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢David Sanchez-Martin博士和Christopher B. Buck博士在项目概念化阶段的评论。我们还要感谢基因组学核心，癌症研究中心，国家癌症研究所，美国国立卫生研究院在初步实验中的帮助，以及协作生物信息学资源，CCR，NCI，NIH在计算分析方面的建议。我们感谢Anna Word女士帮助优化该方法中使用的DNA聚合酶。这项工作利用了NIHHPC生物武夫集群（http://hpc.nih.gov）的计算资源。该项目得到了癌症研究中心，国家癌症研究所，国立卫生研究院的校内计划，NCI主任创新奖（#397172）以及国家癌症研究所的联邦资金的支持，合同号为HHSN261200800001E。我们感谢Tom Misteli，Carol Thiele，Douglas R. Lowy博士以及细胞肿瘤学实验室的所有成员的富有成效的评论。
Materials
| 10x CutSmart buffer | New England BioLabs | B6004 | 10x Digestion buffer |
| 200 proof ethanol | Warner-Graham Company | 200 proof | Ethanol |
| 5-Hydroxymethylcytosine (5-hmC) Monoclonal Antibody [HMC/4D9] | Epigentek | A-1018-100 | Anti-5hmC |
| Acridine Orange/Propidium Iodide Stain | Logos Biosystems | F23001 | Cell counter |
| Acrylamide solution, 40% in H2O, for molecular biology | MilliporeSigma | 01697-500ML | 40% acrylamide solution |
| All-in-One Fluorescence Microscope BZ-X710 | Keyence | BZ-X710 | Scanning microscope |
| Amicon Ultra-0.5 Centrifugal Filter Unit | MilliporeSigma | UFC510024 | Ultrafiltration cassette |
| Ammonium persulfate for molecular biology | MilliporeSigma | A3678-100G | Ammonium persulfate powder |
| Anhydrous DMF | Vector laboratories | S-4001-005 | Anhydrous N,N-dimethylformamide (DMF) |
| Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] | Abcam | ab5408 | Anti-Pol II |
| Anti-TRAP220/MED1 (phospho T1457) antibody | Abcam | ab60950 | Anti-Med1 |
| BciVI | New England BioLabs | R0596L | BciVI |
| Bovine Serum Albumin solution, 20 mg/mL in H2O, low bioburden, protease-free, for molecular biology | MilliporeSigma | B8667-5ML | 20% BSA (Table 7) |
| Bst DNA Polymerase, Large Fragment | New England BioLabs | M0275L | Bst DNA polymerase |
| BT10 Series 10 µl Barrier Tip | NEPTUNE | BT10 | P10 low-retention tip |
| CellCelector | Automated Lab Solutions | N/A | Automated single cell picking robot |
| CellCelector 4 nl nanowell plates for single cell cloning, Plate S200-100 100K, 24 well,ULA | Automated Lab Solutions | CC0079 | 4 nL nanowell plate |
| Chloroform | MilliporeSigma | Chloroform | |
| Corning Costar 96-Well, Cell Culture-Treated, Flat-Bottom Microplate | Corning | 3596 | Flat-bottom 96-well plates |
| Deep Vent (exo-) DNA Polymerase | New England BioLabs | M0259L | Exo– DNA polymerase |
| DNA LoBind Tubes, 0.5 mL | Eppendorf | 30108035 | 0.5 mL DNA low-binding tube |
| DNA Oligo, 1st random primer | Integrated DNA Technologies | N/A, see Table 3 | 1st random primer |
| DNA Oligo, 2nd random primer Cell#01 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#02 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#03 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#04 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#05 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#06 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#07 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#08 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#09 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#10 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#11 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd random primer Cell#12 | Integrated DNA Technologies | N/A, see Table 3 | 2nd random primer |
| DNA Oligo, 2nd synthesis primer | Integrated DNA Technologies | N/A, see Table 3 | 2nd synthesis primer |
| DNA Oligo, Ligation Adaptor | Integrated DNA Technologies | N/A, see Table 3 | Ligation Adaptor |
| DNA Oligo, Reverse Transcription primer | Integrated DNA Technologies | N/A, see Table 3 | Reverse Transcription primer |
| DNase I (RNase-free) | New England BioLabs | M0303L | DNase I (RNase-free, 4 U). |
| DNase I Reaction Buffer | New England BioLabs | B0303S | 10x DNase I buffer (NEB) |
| dNTP Mix (10 mM each) | Thermo Fisher | R0192 | 10 mM dNTPs |
| Fetal Bovine Serum, USA origin, Heat-inactivated | MilliporeSigma | F4135-500ML | Fetal bovine serum |
| HiScribe T7 High Yield RNA Synthesis Kit | New England BioLabs | E2040S | In-vitro-transcription master mix |
| Histone H3K27ac antibody | Active motif | 39133 | Anti-H3K27ac |
| Histone H3K27me3 antibody | Active motif | 39155 | Anti-H3K27me3 |
| IgG from rabbit serum | Millipore Sigma | I5006-10MG | Control IgG |
| Iron oxide(II,III) magnetic nanopowder, 30 nm avg. part. size (TEM), NHS ester functionalized | MilliporeSigma | 747467-1G | NHS ester functionalized 30 nm iron oxide powder |
| K-562 | American Type Culture Collection (ATCC) | CCL-243 | cells |
| Linear Acrylamide (5 mg/mL) | Thermo Fisher | AM9520 | Linear Acrylamide |
| LUNA-FL Dual Fluorescence Cell Counter | Logos Biosystems | L20001 | Cell counter |
| LUNA Cell Counting Slides, 50 Slides | Logos Biosystems | L12001 | Cell counter |
| Mineral oil, BioReagent, for molecular biology, light oil | MilliporeSigma | M5904-500ML | Mineral oil |
| N,N,N′,N′-Tetramethylethylenediamine for molecular biology | MilliporeSigma | T7024-100ML | N,N,N′,N′-Tetramethylethylenediamine |
| NaCl (5 M), RNase-free | Thermo Fisher | AM9760G | 5M NaCl |
| NanoDrop Lite | Thermo Fisher | 2516 | Microvolume spectrophotometer |
| NEST 2 mL 96-Well Deep Well Plate, V Bottom | Opentrons | N/A | 2 mL deep well 96-well plate |
| Non-skirted 96-well PCR plate | Genesee Scientific | 27-405 | 96-well PCR plate |
| NuSive GTG Agarose | Lonza | 50081 | Agarose |
| OmniPur Acrylamide: Bis-acrylamide 19:1, 40% Solution | MilliporeSigma | 1300-500ML | 40%Acrylamide/Bis-acrylamide |
| OT-2 lab robot | Opentrons | OT2 | Automated liquid handling robot |
| Paraformaldehyde, EM Grade, Purified, 20% Aqueous Solution | Electron Microscopy Sciences | 15713 | 20% Pararmaldehyde |
| PBS (10x), pH 7.4 | Thermo Fisher | 70011044 | 10x PBS |
| PIPETMAN Classic P1000 | GILSON | F123602 | A P1000 pipette |
| Protein LoBind Tubes, 1.5 mL | Eppendorf | 925000090 | 1.5 mL Protein low-binding tube |
| QIAgen Gel Extraction kit | Qiagen | 28706 | A P1000 pipette |
| Quant-iT PicoGreen dsDNA Assay | Thermo Fisher | P11495 | dsDNA specific intercalator dye |
| Quick Ligation kit | New England BioLabs | M2200L | T4 DNA ligase (NEB) |
| RNaseOUT Recombinant Ribonuclease Inhibitor | Thermo Fisher | 10777019 | RNAse inhibitor |
| S-4FB Crosslinker (DMF-soluble) | Vector laboratories | S-1004-105 | Succinimidyl 4-formylbenzoate (S-4FB) |
| S-HyNic | Vector laboratories | S-1002-105 | Succinimidyl 6-hydrazinonicotinate acetone hydrazone (S-HyNic) |
| Sodium Acetate, 3 M, pH 5.2, Molecular Biology Grade | MilliporeSigma | 567422-100ML | 3M Sodium acetate (pH 5.2) |
| Sodium bicarbonate, 1M buffer soln., pH 8.5 | Alfa Aesar | J60408 | 1M sodium bicarbonate buffer, pH 8.5 |
| Sodium phosphate dibasic for molecular biology | MilliporeSigma | S3264-250G | Na2HPO4 |
| Sodium phosphate monobasic for molecular biology | MilliporeSigma | S3139-250G | NaH2PO4 |
| SuperScript IV reverse transcriptase | Thermo Fisher | 18090050 | Reverse transcriptase |
| SYBR Gold Nucleic Acid Gel Stain (10,000x Concentrate in DMSO) | Thermo Fisher | S11494 | An intercalator dye for DNA |
| T4 DNA Ligase Reaction Buffer | New England BioLabs | B0202S | 10x T4 DNA ligase reaction buffer |
| ThermoPol Reaction Buffer Pack | New England BioLabs | B9004S | 10x TPM-T buffer (Tris-HCl/Pottasium chloride/Magnesium sulfate/Triton X-100) |
| TRIzol LS reagent | Thermo Fisher | 10296-028 | Guanidinium thiocyanate-phenol-chloroform extraction |
| TruSeq Nano DNA library prep kit | Illumina | 20015965 | A DNA library preparation kit (see also the manufacturer's instruction) |
| Ultramer DNA Oligo, Anti-5hmC_Ab#005 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Anti-H3K27ac_Ab#002 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Anti-H3K27me3_Ab#003 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Anti-Med1_Ab#004 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Anti-Pol II_Ab#006 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for antibody |
| Ultramer DNA Oligo, Control IgG_Ab#001 | Integrated DNA Technologies | N/A, see Table 3 | An amine-modified DNA probe for control IgG |
| UltraPure 0.5 M EDTA, pH 8.0 | Thermo Fisher | 15575020 | 0.5M EDTA, pH 8.0 |
| UltraPure DNase/RNase-Free Distilled Water | Thermo Fisher | 10977023 | Ultrapure water |
| Zeba Splin Desalting Columns, 7K MWCO, 0.5 mL | Thermo Fisher | 89882 | Desalting column |
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Cite This Article
Ohnuki, H., Venzon, D. J., Lobanov, A., Tosato, G. Reusable Single Cell for Iterative Epigenomic Analyses. J. Vis. Exp. (180), e63456, doi:10.3791/63456 (2022).