白色念珠菌中转录因子-DNA结合相互作用的全基因组分析：全面的CUT&RUN方法和数据分析工作流程
Summary
该协议描述了一种实验方法和数据分析工作流程，用于在靶标下切割并使用核酸酶（CUT&RUN）在人类真菌病原体 白色念珠菌中释放。
Abstract
调节转录因子控制许多重要的生物过程，包括细胞分化，对环境扰动和压力的反应以及宿主 – 病原体相互作用。确定调节转录因子与DNA的全基因组结合对于了解转录因子在这些通常复杂的生物过程中的功能至关重要。使用核酸酶（CUT&RUN）在靶标下切割和释放是一种用于 体内 蛋白质 – DNA结合相互作用的全基因组图谱的现代方法，是传统和广泛使用的染色质免疫沉淀随后测序（ChIP-seq）方法的有吸引力的替代方案。CUT&RUN适用于更高通量的实验设置，并且具有比ChIP-seq更高的动态范围和更低的每样本测序成本。在这里，描述了一个全面的CUT&RUN方案和随附的数据分析工作流程，这些工作流程是为全基因组分析人类真菌病原体 白色念珠菌 中的转录因子 – DNA结合相互作用而量身定制的。这个详细的方案包括所有必要的实验程序，从转录因子编码基因的表位标记到测序的文库准备;此外，它还包括用于CUT&RUN数据分析的定制计算工作流程。
Introduction
白色念珠菌 是一种临床相关的多态性人类真菌病原体，以各种不同的生长模式存在，例如浮游（自由浮动）生长模式和作为由细胞外基质保护的紧密粘附的细胞群落，称为生物膜生长模式1，2，3。与其他发育和细胞过程类似，生物膜发育是白色 念珠菌 的一种重要的毒力性状，已知其在转录水平上由以序列特异性方式与DNA结合的调节转录因子（TFs）控制4。最近，染色质调节剂和组蛋白修饰剂也成为通过介导DNA可及性成为 白色念珠菌 生物膜形成5 和形态发生6 的重要调节剂。为了了解这种重要真菌病原体的复杂生物学，在不同的发育和细胞过程中确定特定TF的全基因组定位的有效方法是有价值的。
染色质免疫沉淀后测序（ChIP-seq）是研究白色念珠菌5，6中蛋白质 – DNA相互作用的广泛方法，并且在很大程度上取代了更经典的染色质免疫沉淀，然后是微阵列（ChIP-chip）9方法。然而，ChIP-seq和ChIP芯片方法都需要大量的输入细胞10，这在特定样品和生长模式的背景下研究TF时可能是一个复杂的因素，例如从患者收集的生物膜或感染的动物模型。此外，染色质免疫沉淀（ChIP）测定通常在整个基因组中产生大量的背景信号，需要高水平的富集以使目标靶标充分分离信号与噪声。虽然ChIP芯片测定在今天基本上已经过时了，但ChIP-seq所需的测序深度使得这种测定对于许多研究人员来说过于昂贵，特别是那些研究多种TF和/或染色质相关蛋白的研究人员。
使用核酸酶（CUT&RUN）在靶标下切割和释放是ChIP-seq的有吸引力的替代品。它由Henikof实验室于2017年开发，以规避ChIP-seq和染色质内源性裂解的局限性，然后对ChEC-seq11，12进行测序，这是另一种在全基因组水平上鉴定蛋白质 – DNA相互作用的方法，同时提供TF和染色质相关蛋白质的高分辨率，全基因组图谱13.CUT&RUN依赖于使用系留微球菌核酸酶在透化核内靶向消化染色质，然后对消化的DNA片段9，10进行测序。由于DNA片段是在由目标蛋白质结合的位点特异性生成的，而不是像ChIP测定那样通过随机片段化在整个基因组中产生，因此CUT&RUN方法导致背景信号大大减少，因此，与ChIP-seq 11，13相比，需要测序深度的1/10，14.这些改进最终导致测序成本的显着降低，并减少了每个样品作为起始材料所需的输入细胞总数。
在这里，描述了一个强大的CUT&RUN方案，该方案已经过调整和优化，用于确定从生物膜和浮游培养物中分离的 白色念珠菌 细胞中TF的全基因组定位。还提供了一个全面的数据分析管道，该管道可以处理和分析生成的序列数据，并要求用户在编码或生物信息学方面具有最少的专业知识。简而言之，该协议描述了TF编码基因的表位标记，生物膜和浮游细胞的收获，完整透化核的分离，针对特定蛋白质或表位标记的蛋白质的一抗孵育，嵌合A / G-微球菌核酸酶（pAG-MNase）融合蛋白与一抗的系留，染色质消化后的基因组DNA恢复以及用于测序的基因组DNA文库的制备。
实验性的CUT&RUN协议之后是一个专门构建的数据分析管道，该管道以FASTQ格式进行原始DNA测序读取，并实施所有必需的处理步骤，以提供由感兴趣的TF（由一抗靶向）结合的显着富集位点的完整列表。请注意，所描述的文库制备方案的多个步骤已针对TF（而不是核小体）的CUT&RUN分析进行了专门调整和优化。虽然本手稿中提供的数据是使用商业CUT&RUN试剂盒的TF特异性改编生成的，但这些方案也已使用单独来源的组分（即pAG-MNase酶和磁性DNA纯化珠）和内部制备的缓冲液进行了验证，这可以显着降低实验成本。下面以分步形式详细介绍了全面的实验和数据分析协议。所有试剂和关键设备以及缓冲液和培养基配方分别列在 材料表 和 补充文件1中。
Protocol
1. 白色 念珠菌菌株的表位标记 将目的基因及其1 kb上游和下游侧翼序列从 念珠菌 基因组数据库上传到引物设计工具（参见 材料表）。通过突出显示终止密码子的上游和下游50 bp来设计指导RNA（gRNA），然后单击右侧的 gRNA选择 工具。选择“ 设计和分析参考线”。使用 Ca22（白色念珠菌 SC5314 组装 22（二倍体））基因组?…
Representative Results
这种强大的CUT&RUN方案经过调整和优化，用于研究 白色念珠菌 生物膜和浮游培养物中特定TF的全基因组定位（有关实验方法的概述，请参见 图2 ）。还包括一个全面的数据分析管道，以促进对生成的CUT&RUN测序数据的分析，并要求用户在编码或生物信息学方面具有最少的专业知识（有关分析管道的概述，请参见 图3 ）。与ChIP芯片和ChIP-seq方法相反?…
Discussion
该协议为白色念珠 菌中调节性TF的全基因组定位提供了全面的实验和计算管道。它被设计为任何接受过标准微生物学和分子生物学培训的人都可以轻松访问。通过利用CUT&RUN测定的高动态范围和低样品输入要求，并包括对 白色念珠菌 生物膜和浮游培养物中TF-DNA结合相互作用的定位的优化，该协议为传统的ChIP-seq方法提供了一种强大且低成本的替代方案。与ChIP-seq相比，CUT&RUN的灵敏度?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢Nobile和Hernday实验室的所有过去和现在的成员对手稿的反馈。这项工作得到了美国国立卫生研究院（NIH）国家普通医学科学研究所（NIGMS）奖项R35GM124594的支持，并由Kamangar家族以C.J.N.捐赠的椅子的形式提供支持。这项工作还得到了NIH国家过敏和传染病研究所（NIAID）的支持，奖项编号为R15AI137975，以A.D.H.C.L.E.得到了NIH国家牙科和颅面研究所（NIDCR）奖学金F31DE028488的支持。内容由作者自行负责，不代表资助者的观点。资助者在研究的设计中没有发挥作用;收集、分析或解释数据;在手稿的写作中;或在决定公布结果。
Materials
| 0.22 μm filter | Millipore Sigma | SLGPM33RS | |
| 0.65 mL low-adhesion tubes | VWR | 490003-190 | |
| 1 M CaCl2 | Fisher Scientific | 50-152-341 | |
| 1 M PIPES | Fisher Scientific | AAJ61224AK | |
| 12-well untreated cell culture plates | Corning | 351143 | |
| 2-mercaptoethanol | Sigma-Aldrich | 60-24-2 | |
| 2% Digitonin | Fisher Scientific | CHR103MI | |
| 50 mL conical tubes | VWR | 89039-658 | |
| 5x phusion HF buffer | Fisher Scientific | F530S | Item part of the Phusion high fidelity DNA polymerase; referred to in text as "DNA polymerase buffer" |
| Agar | Criterion | C5001 | |
| Agencourt AMPure XP magnetic beads | Beckman Coulter | A63880 | |
| Agilent Bioanalyzer | Agilent | G2939BA | Referred to in the text as "capillary electrophoresis instrument"; user-dependepent |
| Amplitube PCR reaction strips with attached caps, Simport Scientific | VWR | 89133-910 | |
| Bacto peptone | BD Biosciences | 211677 | |
| Benchling primer design tool | Benchling | https://www.benchling.com/molecular-biology/; Referred to in the text as "the primer design tool" | |
| Betaine | Fisher Scientific | AAJ77507AB | |
| Calcofluor white stain | Sigma-Aldrich | 18909-100ML-F | |
| Candida Genome Database | http://www.candidagenome.org/ | ||
| Concanavalin A (ConA) conjugated paramagnetic beads | Polysciences | 86057-3 | |
| Conda software | https://docs.conda.io/en/latest/miniconda.html | ||
| curl tool | http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly21/current/C_albicans_SC5314_A21_current_ chromosomes.fasta.gz |
||
| CUTANA ChIC/CUT&RUN kit | Epicypher | 14-1048 | Referred to in the text as "the CUT&RUN kit" |
| Deoxynucleotide (dNTP) solution mix (10 mM) | New England Biolabs | N0447S | |
| Dextrose (D-glucose) | Fisher Scientific | D163 | |
| Difco D-mannitol | BD Biosciences | 217020 | |
| Disposable cuvettes | Fisher Scientific | 14-955-127 | |
| Disposable transfer pipets | Fisher Scientific | 13-711-20 | |
| DNA Gel Loading Dye (6x) | Fisher Scientific | R0611 | |
| DreamTaq green DNA polymerase | Fisher Scientific | EP0713 | Referred to in the text as "cPCR DNA polymerase" |
| DreamTaq green DNA polymerase buffer | Fisher Scientific | EP0713 | Item part of the DreamTaq green DNA polymerase; referred to in the text as "cPCR DNA polymerase buffer" |
| E. coli spike-in DNA | Epicypher | 18-1401 | |
| ELMI Microplate incubator | ELMI | TRMS-04 | Referred to in the text as "microplate incubator" |
| End Prep Enzyme Mix | Item part of the NEBNext Ultra II DNA Library Prep kit | ||
| End Prep Reaction Buffer | Item part of the NEBNext Ultra II DNA Library Prep kit | ||
| Ethanol 200 proof | VWR | 89125-170 | |
| FastDigest MssI | Fisher Scientific | FD1344 | Referred to in the text as "restriction enzyme" |
| FastDigest MssI Buffer | Fisher Scientific | FD1344 | Item part of the FastDigest MssI kit; referred to in the text as "restriction enzyme buffer" |
| Ficoll 400 | Fisher BioReagents | BP525-25 | |
| Fluorescence microscope | User-dependent | ||
| Gel electrophoresis apparatus | User-dependent | ||
| GeneRuler low range DNA ladder | Fisher Scientific | FERSM1192 | |
| GitBash workflow | https://gitforwindows.org/ | ||
| GitHub source code | https://github.com/akshayparopkari/cut_run_analysis | ||
| HEPES-KOH pH 7.5 | Boston BioProducts | BBH-75-K | |
| High-speed centrifuge | User-dependent | ||
| Isopropanol | Sigma-Aldrich | PX1830-4 | |
| Lens paper | VWR | 52846-001 | |
| Ligation Enhancer | Item part of the NEBNext Ultra II DNA Library Prep kit | ||
| Lithium acetate dihydrate | MP Biomedicals | 215525683 | |
| Living Colors Full-Length GFP polyclonal antibody | Takara | 632592 | User-dependent |
| MACS2 | https://pypi.org/project/MACS2/ | ||
| Magnetic separation rack, 0.2 mL tubes | Epicypher | 10-0008 | |
| Magnetic separation rack, 1.5 mL tubes | Fisher Scientific | MR02 | |
| MgCl2 | Sigma-Aldrich | M8266 | |
| Microcentrifuge tubes 1.5 mL | Fisher Scientific | 05-408-129 | |
| Microplate and cuvette spectrophotometer | BioTek | EPOCH2TC | Referred to in the text as "spectrophotometer"; user-dependent |
| MochiView | http://www.johnsonlab.ucsf.edu/mochiview-downloads | ||
| MOPS | Sigma-Aldrich | M3183 | |
| NaCl | VWR | 470302-522 | |
| NaOH | Fisher Scientific | S318-500 | |
| NCBI GEO | https://www.ncbi.nlm.nih.gov/geo/ | ||
| NEBNext Adaptor for Illumina | Item part of the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1); referred to in the text as "Adapter" | ||
| NEBNext Index X Primer for Illumina | Item part of the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1); referred to in the text as "Reverse Uniquely Indexed Library Amplification Primer" | ||
| NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) | New England Biolabs | E7335S | |
| NEBNext Ultra II DNA Library Prep kit | New England Biolabs | E7645S | Referred to in the text as "library prep kit" |
| NEBNext Universal PCR Primer for Illumina | Item part of the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1); referred to in the text as "Universal Forward Library Amplification Primer" | ||
| Nourseothricin sulfate (NAT) | Goldbio | N-500-2 | |
| Novex TBE Gels, 10%, 15 well | Fisher Scientific | EC62755BOX | |
| Nutating mixer | VWR | 82007-202 | |
| Nutrient broth | Criterion | C6471 | |
| pADH110 | Addgene | 90982 | Referred to in the text as "plasmid repository ID# 90982" |
| pADH119 | Addgene | 90985 | Referred to in the text as "plasmid repository ID# 90985" |
| pADH137 | Addgene | 90986 | Referred to in the text as "plasmid repository ID# 90986" |
| pADH139 | Addgene | 90987 | Referred to in the text as "plasmid repository ID# 90987" |
| pADH140 | Addgene | 90988 | Referred to in the text as "plasmid repository ID# 90988" |
| pAG-MNase | Epicypher | 15-1016 or 15-1116 | 50 rxn or 250 rxn |
| pCE1 | Addgene | 174434 | Referred to in the text as "plasmid repository ID# 174434" |
| Petri dishes with clear lid | Fisher Scientific | FB0875712 | |
| Phusion high fidelity DNA polymerase | Fisher Scientific | F530S | Referred to in the text as "DNA polymerase" |
| Polyethylene glycol (PEG) 3350 | VWR | 10791-816 | |
| Potassium phosphate monobasic | Fisher Scientific | P285-500 | |
| Qubit 1x dsDNA HS assay kit | Invitrogen | Q33230 | |
| Qubit fluorometer | Life Technologies | Q33216 | Referred to in the text as "fluorometer"; user-dependent |
| Rabbit IgG negative control antibody | Epicypher | 13-0042 | |
| RNase A | Sigma-Aldrich | 10109169001 | |
| Roche Complete protease inhibitor (EDTA-free) tablets | Sigma-Aldrich | 5056489001 | |
| RPMI-1640 | Sigma-Aldrich | R6504 | |
| Shaking incubator | Eppendorf | M12820004 | User-dependent |
| Sorbitol | Sigma-Aldrich | S1876-500G | |
| Spin-X centrifuge tube filters | Fisher Scientific | 07-200-385 | |
| Sterile inoculating loops | VWR | 30002-094 | |
| SYBR Gold nucleic acid gel stain | Fisher Scientific | S11494 | |
| SYTO 13 nucleic acid stain | Fisher Scientific | S7575 | Referred to in the text as "nucleic acid gel stain" |
| Thermocycler | User-dependent | ||
| ThermoMixer C | Eppendorf | 5382000023 | |
| Tris (hydroxymethyl) aminomethane | Sigma-Aldrich | 252859-100G | |
| Ultra II Ligation Master Mix | Item part of the NEBNext Ultra II DNA Library Prep kit; referred to in the text as "Ligation Master Mix" | ||
| Ultra II Q5 Master Mix | Item part of the NEBNext Ultra II DNA Library Prep kit; referred to in the text as "High Fidelity DNA Polymerase Master Mix " | ||
| UltraPure salmon sperm DNA solution | Invitrogen | 15632011 | |
| USER Enzyme | Item part of the NEBNext Ultra II DNA Library Prep kit; referred to in the text as "Uracil Excision Enzyme" | ||
| Vortex mixer | VWR | 10153-834 | |
| wget tool | http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly21/current/C_albicans_SC5314_A21_current_ chromosomes.fasta.gz |
||
| Yeast extract | Criterion | C7341 | |
| Zymolyase 100T (lyticase, yeast lytic enzyme) | Fisher Scientific | NC0439194 |
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Citar este artigo
Qasim, M. N., Valle Arevalo, A., Paropkari, A. D., Ennis, C. L., Sindi, S. S., Nobile, C. J., Hernday, A. D. Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow. J. Vis. Exp. (182), e63655, doi:10.3791/63655 (2022).