方法文章

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT&RUN Method and Data Analysis Workflow

DOI：

10.3791/63655

⸱

2022年4月1日

 ,  ,  ,  ,  ,  , 
1Department of Molecular and Cell Biology, University of California, Merced, 2Quantitative and Systems Biology Graduate Program, University of California, Merced, 3Department of Applied Mathematics, University of California, Merced, 4Health Sciences Research Institute, University of California, Merced

* These authors contributed equally

本文内容

摘要

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This protocol describes an experimental method and data analysis workflow for cleavage under targets and release using nuclease (CUT&RUN) in the human fungal pathogen Candida albicans.

摘要

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Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations and stresses, and host-pathogen interactions. Determining the genome-wide binding of regulatory transcription factors to DNA is essential to understanding the function of transcription factors in these often complex biological processes. Cleavage under targets and release using nuclease (CUT&RUN) is a modern method for genome-wide mapping of in vivo protein-DNA binding interactions that is an attractive alternative to the traditional and widely used chromatin immunoprecipitation followed by sequencing (ChIP-seq) method. CUT&RUN is amenable to a higher-throughput experimental setup and has a substantially higher dynamic range with lower per-sample sequencing costs than ChIP-seq. Here, a comprehensive CUT&RUN protocol and accompanying data analysis workflow tailored for genome-wide analysis of transcription factor-DNA binding interactions in the human fungal pathogen Candida albicans are described. This detailed protocol includes all necessary experimental procedures, from epitope tagging of transcription factor-coding genes to library preparation for sequencing; additionally, it includes a customized computational workflow for CUT&RUN data analysis.

引言

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Candida albicans is a clinically relevant, polymorphic human fungal pathogen that exists in a variety of different modes of growth, such as the planktonic (free-floating) mode of growth and as communities of tightly adhered cells protected by an extracellular matrix, known as the biofilm mode of growth1,2,3. Similar to other developmental and cellular processes, biofilm development is an important C. albicans virulence trait that is known to be controlled at the transcriptional level by regulatory transcription factors (TFs) that bind to DNA in a sequence-sp....

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方案

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1. Epitope Tagging of C. albicans strains

  1. Upload the gene of interest, along with its 1 kb upstream and downstream flanking sequences, from the Candida Genome Database to the primer design tool (see the Table of Materials). Design a guide RNA (gRNA) by highlighting 50 bp upstream and downstream from the stop codon, and click the gRNA selection tool on the right. Select Design and Analyze Guides. Use the Ca22 (Candida albicans SC5314 Assembly 22 (diploid)) genome and an NGG (SpCas9, 3' side) protospacer adjacent motif (PAM) for the guide parameters, and click ....

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结果

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This robust CUT&RUN protocol was adapted and optimized for investigating the genome-wide localization of specific TFs in C. albicans biofilms and planktonic cultures (see Figure 2 for an overview of the experimental approach). A thorough data analysis pipeline is also included to facilitate analysis of the resulting CUT&RUN sequencing data and requires users to have minimal expertise in coding or bioinformatics (see Figure 3 for an overview of t.......

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讨论

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This protocol presents a comprehensive experimental and computational pipeline for genome-wide localization of regulatory TFs in C. albicans. It is designed to be highly accessible to anyone with standard microbiology and molecular biology training. By leveraging the high dynamic range and low sample input requirements of the CUT&RUN assay and including optimizations for the localization of TF-DNA binding interactions in C. albicans biofilm and planktonic cultures, this protocol presents a powerful .......

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披露

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Clarissa J. Nobile is a cofounder of BioSynesis, Inc., a company developing diagnostics and therapeutics for biofilm infections.

致谢

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We thank all past and present members of the Nobile and Hernday laboratories for feedback on the manuscript. This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) award number R35GM124594 and by the Kamangar family in the form of an endowed chair to C.J.N. This work was also supported by the NIH National Institute of Allergy and Infectious Diseases (NIAID) award number R15AI137975 to A.D.H. C.L.E. was supported by the NIH National Institute of Dental and Craniofacial Research (NIDCR) fellowship number F31DE028488. The content is the sole responsibility of the authors and does not represent the vi....

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材料

本文使用的材料清单
姓名公司目录编号评论
0.22 μm 过滤器Millipore SigmaSLGPM33RS
0.65 mL 低粘附管VWR490003-190
1 M CaCl2Fisher Scientific50-152-341
1 M 管道Fisher ScientificAAJ61224AK
12 孔未经处理的细胞培养板Corning351143
2-巯基乙醇Sigma-Aldrich60-24-2
2% 洋地黄皂Fisher ScientificCHR103MI
50 mL 锥形管VWR89039-658
5x phusion HF 缓冲液Fisher ScientificF530SPhusion 高保真 DNA 聚合酶的一部分;在文中称为"DNA 聚合酶缓冲液"
琼脂标准C5001
Agencourt AMPure XP 磁珠Beckman CoulterA63880
安捷伦生物分析仪安捷伦G2939BA在文中称为"毛细管电泳仪";用户依赖
Amplitube PCR 反应条，带连接盖，Simport ScientificVWR89133-910
Bacto 蛋白胨BD Biosciences211677
Benchling 底漆设计工具Benchlinghttps://www.benchling.com/molecular-biology/;在文中称为"引物设计工具"
甜菜碱费舍尔科学AAJ77507AB
钙荧光白色染色Sigma-Aldrich18909-100ML-F
念珠菌 基因组数据库http://www.candidagenome.org/
伴刀豆球蛋白 A （ConA） 共轭顺磁性珠Polysciences 86057-3
Conda 软件https://docs.conda.io/en/latest/miniconda.html
卷曲工具http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly21/current/C_albicans_SC5314_A21_current_
chromosomes.fasta.gz
CUTANA ChIC/CUT&RUN 套件Epicypher14-1048在文本中称为"CUT&RUN 试剂盒"
脱氧核苷酸 （dNTP） 溶液混合物 （10 mM）New England BiolabsN0447S
葡萄糖 （D-葡萄糖）Fisher ScientificD163
Difco D-甘露醇 BD Biosciences217020
一次性比色皿Fisher Scientific14-955-127
一次性移液管Fisher Scientific13-711-20
DNA 凝胶上样染料 （6x）Fisher ScientificR0611
DreamTaq 绿色 DNA 聚合酶Fisher ScientificEP0713在文中称为"cPCR DNA 聚合酶"
DreamTaq green DNA聚合酶缓冲液Fisher ScientificEP0713DreamTaq green DNA聚合酶的一部分;文中称为"cPCR DNA聚合酶缓冲液"
E. coli spike-in DNAEpicypher18-1401
ELMI微孔板培养箱ELMITRMS-04文中称为"微孔板培养箱"
结束制备酶混合物NEBNext Ultra II DNA 文库制备试剂盒的一部分
结束制备反应缓冲液NEBNext Ultra II DNA 文库制备试剂盒的一部分
乙醇 200 证明VWR89125-170
FastDigest MssIFisher ScientificFD1344在文中称为"限制性内切酶"
FastDigest MssI 缓冲液Fisher科学级FD1344FastDigest MssI 试剂盒的项目部分;在文中称为"限制性内切酶缓冲液"
Ficoll 400Fisher BioReagentsBP525-25
荧光显微镜用户依赖性
凝胶电泳仪用户依赖
GeneRuler 低范围 DNA 分子量标准Fisher Scientific
GitBash 工作流程https://gitforwindows.org/
GitHub 源代码https://github.com/akshayparopkari/cut_run_analysis
HEPES-KOH pH 7.5Boston BioProductsBBH-75-K
高速离心机用户依赖
异丙醇Sigma-AldrichPX1830-4
镜头纸VWR52846-001
连接增强剂项目 NEBNext Ultra II DNA 文库制备试剂盒的一部分
乙酸锂二水合MP Biomedicals215525683
Living Colors 全长 GFP 多克隆抗体Takara632592用户依赖
MACS2https://pypi.org/project/MACS2/
磁性分离架，0.2 mL 管Epicypher10-0008
磁性分离架，1.5 mL 管Fisher ScientificMR02
MgCl2Sigma-AldrichM8266
微量离心管 1.5 mLFisher Scientific05-408-129
微孔板和比色皿分光光度计BioTekEPOCH2TC在文中称为"分光光度计";取决于用户
MochiViewhttp://www.johnsonlab.ucsf.edu/mochiview-downloads
MOPSSigma-AldrichM3183
NaClVWR470302-522
NaOHFisher ScientificS318-500
NCBI GEOhttps://www.ncbi.nlm.nih.gov/geo/
NEBNext Adaptor for IlluminaItem 是 NEBNext Multiplex Oligos for Illumina （Index Primers Set 1） 的一部分;文中称为"Adapter"
NEBNext Index X Primer for IlluminaItem 是 NEBNext Multiplex Oligos for Illumina （Index Primers Set 1） 的一部分;文中称为"Reverse Unique， Indexed Library扩增引物"
NEBNext Multiplex Oligos for Illumina （Index Primers Set 1）New England BiolabsE7335S
NEBNext Ultra II DNA 文库制备试剂盒New England BiolabsE7645S在文中称为"文库制备试剂盒"
NEBNext Universal PCR Primer for IlluminaNEBNext Multiplex Oligos for Illumina 的项目部分（索引引物组 1）;文中称为"通用正向文库扩增引物"
Nourseothricin sulfate （NAT）GoldbioN-500-2
Novex TBE 凝胶，10%，15 孔Fisher ScientificEC62755BOX
章动混合器VWR82007-202
营养液汤标准C6471
pADH110Addgene90982中称为"质粒库 ID# 90982"
pADH119Addgene90985中称为"质粒库 ID# 90985"
pADH137Addgene90986中称为"质粒库 ID# 90986"
pADH139Addgene90987文中称为"质粒库 ID# 90987"
pADH140Addgene90988在文中称为"质粒库 ID# 90988"
pAG-MNaseEpicypher15-1016 或 15-111650 rxn 或 250 rxn
pCE1Addgene174434在文中称为"质粒库 ID# 174434"
带透明盖的培养皿Fisher ScientificFB0875712
Phusion 高保真 DNA 聚合酶Fisher ScientificF530S文中称为"DNA 聚合酶"
乙二醇 （PEG） 3350VWR10791-816
磷酸二氢钾Fisher ScientificP285-500
Qubit 1x dsDNA HS 检测试剂盒InvitrogenQ33230
Qubit 荧光计Life TechnologiesQ33216在文中称为"荧光计";用户依赖
兔 IgG 阴性对照抗体Epicypher13-0042
RNase ASigma-Aldrich10109169001
Roche 完全蛋白酶抑制剂（不含 EDTA）片Sigma-Aldrich5056489001
RPMI-1640Sigma-AldrichR6504
振荡培养箱EppendorfM12820004用户依赖
性山梨糖醇Sigma-AldrichS1876-500G
Spin-X 离心管过滤器Fisher Scientific07-200-385
无菌接种环VWR30002-094
SYBR Gold 核酸凝胶染料Fisher ScientificS11494
SYTO 13 核酸染色Fisher ScientificS7575文中称为"核酸凝胶染色剂"
热循环仪用户依赖
ThermoMixer CEppendorf5382000023
Tris（羟甲基）氨基甲烷Sigma-Aldrich252859-100G
Ultra II 连接预混液NEBNext Ultra II DNA 文库制备试剂盒的项目部分;文中称为"连接预混液"
Ultra II Q5 预混液项目 NEBNext Ultra II DNA 文库制备试剂盒的一部分;文中称为"高保真 DNA 聚合酶预混液"
UltraPure 鲑鱼精子 DNA 溶液Invitrogen15632011
USER 酶NEBNext Ultra II DNA 文库制备试剂盒的项目部分;文中简称为"尿嘧啶切除酶"
涡旋混合器VWR10153-834
wget toolhttp://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly21/current/C_albicans_SC5314_A21_current_
chromosomes.fasta.gz
酵母提取物CriterionC7341
酶解酶 100T（溶壁酶，酵母裂解酶）Fisher ScientificNC0439194
苷 的 剂 性 FERSM1192性物 性 文 文 文聚性剂 剂 型

参考文献

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CUT And Run DNA

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