Name: Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT and RUN Method and Data Analysis Workflow (Video) | JoVE Journal
Uploaded: 2022-04-01T14:45:00.000+00:00
Duration: 7 min 48 s
Description: Learn CUT&RUN for genome-wide profiling of transcription factor-DNA binding in Candida albicans. This computational workflow offers faster, more sensitive analysis than traditional ChIP methods for pathogen research.

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We thank all past and present members of the Nobile and Hernday laboratories for feedback on the manuscript.&#160;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 views of the funders. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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<li>Lohse, M. B., Gulati, M., Johnson, A. D., Nobile, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+regulation+of+single-and+multi-species+Candida+albicans+biofilms%5BTitle%5D)+AND+%22Nature+Reviews+Microbiology%22%5BJournal%5D)">Development and regulation of single-and multi-species Candida albicans biofilms.</a> Nature Reviews Microbiology. 16 (1), 19-31 (2018).</li>
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<li>Nobile, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+recently+evolved+transcriptional+network+controls+biofilm+development+in+Candida+albicans%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A recently evolved transcriptional network controls biofilm development in Candida albicans.</a> Cell. 148 (1-2), 126-138 (2012).</li>
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<li>Qasim, M. N., Valle Arevalo, A., Nobile, C. J., Hernday, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+roles+of+chromatin+accessibility+in+regulating+the+Candida+albicans+white-opaque+phenotypic+switch%5BTitle%5D)+AND+%22Journal+of+Fungi%22%5BJournal%5D)">The roles of chromatin accessibility in regulating the Candida albicans white-opaque phenotypic switch.</a> Journal of Fungi. 7 (1), 37(2021).</li>
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<li>Lohse, M. B., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+Wor4%2C+a+new+transcriptional+regulator+of+white-opaque+switching%5BTitle%5D)+AND+%22G3%3A+Genes%2C+Genomes%2C+Genetics%22%5BJournal%5D)">Identification and characterization of Wor4, a new transcriptional regulator of white-opaque switching.</a> G3: Genes, Genomes, Genetics. 6 (3), 721-729 (2016).</li>
<li>Hernday, A. D., Noble, S. M., Mitrovich, Q. M., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+and+molecular+biology+in+Candida+albicans%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Genetics and molecular biology in Candida albicans.</a> Methods in Enzymology. 470, 737-758 (2010).</li>
<li>Lee, T. I., Johnstone, S. E., Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+immunoprecipitation+and+microarray-based+analysis+of+protein+location%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Chromatin immunoprecipitation and microarray-based analysis of protein location.</a> Nature Protocols. 1 (2), 729-748 (2006).</li>
<li>Zentner, G. E., Kasinathan, S., Xin, B., Rohs, R., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ChEC-seq+kinetics+discriminates+transcription+factor+binding+sites+by+DNA+sequence+and+shape+in+vivo%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.</a> Nature Communications. 6, 8733(2015).</li>
<li>Schmid, M., Durussel, T., Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ChIC+and+ChEC%3A+Genomic+mapping+of+chromatin+proteins%5BTitle%5D)+AND+%22Molecular+Cell%22%5BJournal%5D)">ChIC and ChEC: Genomic mapping of chromatin proteins.</a> Molecular Cell. 16 (1), 147-157 (2004).</li>
<li>Skene, P. J., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+efficient+targeted+nuclease+strategy+for+high-resolution+mapping+of+DNA+binding+sites%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.</a> eLife. 6, 21856(2017).</li>
<li>Skene, P. J., Henikoff, J. G., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+in+situ+genome-wide+profiling+with+high+efficiency+for+low+cell+numbers%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Targeted in situ genome-wide profiling with high efficiency for low cell numbers.</a> Nature Protocols. 13 (5), 1006-1019 (2018).</li>
<li>Nguyen, N., Quail, M. M. F., Hernday, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+efficient%2C+rapid%2C+and+recyclable+system+for+CRISPR-mediated+genome+editing+in+Candida+albicans%5BTitle%5D)+AND+%22American+Society+For+Microbiology%22%5BJournal%5D)">An efficient, rapid, and recyclable system for CRISPR-mediated genome editing in Candida albicans.</a> American Society For Microbiology. 2 (2), 00149(2017).</li>
<li>Ennis, C. L., Hernday, A. D., Nobile, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+markerless+CRISPR-mediated+system+for+genome+editing+in+Candida+auris+reveals+a+conserved+role+for+Cas5+in+the+caspofungin+response%5BTitle%5D)+AND+%22Microbiology+Spectrum%22%5BJournal%5D)">A markerless CRISPR-mediated system for genome editing in Candida auris reveals a conserved role for Cas5 in the caspofungin response.</a> Microbiology Spectrum. 9 (3), 0182021(2021).</li>
<li>Meers, M. P., Bryson, T. D., Henikoff, J. G., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+CUT%26RUN+chromatin+profiling+tools%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Improved CUT&RUN chromatin profiling tools.</a> eLife. 8, 46314(2019).</li>
<li>Skrzypek, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Candida+Genome+Database+%28CGD%29%3A+Incorporation+of+Assembly+22%2C+systematic+identifiers+and+visualization+of+high+throughput+sequencing+data%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">The Candida Genome Database (CGD): Incorporation of Assembly 22, systematic identifiers and visualization of high throughput sequencing data.</a> Nucleic Acids Research. 45, 592-596 (2017).</li>
<li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BEDTools%3A+a+flexible+suite+of+utilities+for+comparing+genomic+features%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26 (6), 841-842 (2010).</li>
<li>Landt, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ChIP-seq+guidelines+and+practices+of+the+ENCODE+and+modENCODE+consortia%5BTitle%5D)+AND+%22Genome+Research%22%5BJournal%5D)">ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.</a> Genome Research. 22 (9), 1813-1831 (2012).</li>
<li>Boyd, J., Rodriguez, P., Schjerven, H., Frietze, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ssvQC%3A+an+integrated+CUT%26RUN+quality+control+workflow+for+histone+modifications+and+transcription+factors%5BTitle%5D)+AND+%22BMC+Research+Notes%22%5BJournal%5D)">ssvQC: an integrated CUT&RUN quality control workflow for histone modifications and transcription factors.</a> BMC Research Notes. 14 (1), 366(2021).</li>
<li>Kent, W. J., Zweig, A. S., Barber, G., Hinrichs, A. S., Karolchik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BigWig+and+BigBed%3A+enabling+browsing+of+large+distributed+datasets%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">BigWig and BigBed: enabling browsing of large distributed datasets.</a> Bioinformatics. 26 (17), 2204-2207 (2010).</li>
<li>Homann, O. R., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MochiView%3A+Versatile+software+for+genome+browsing+and+DNA+motif+analysis%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">MochiView: Versatile software for genome browsing and DNA motif analysis.</a> BMC Biology. 8, 49(2010).</li>
<li>Hainer, S. J., Fazzio, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+chromatin+profiling+using+CUT%26RUN%5BTitle%5D)+AND+%22Current+Protocols+in+Molecular+Biology%22%5BJournal%5D)">High-resolution chromatin profiling using CUT&RUN.</a> Current Protocols in Molecular Biology. 126 (1), 85(2019).</li>
<li>Kaya-Okur, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CUT%26Tag+for+efficient+epigenomic+profiling+of+small+samples+and+single+cells%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">CUT&Tag for efficient epigenomic profiling of small samples and single cells.</a> Nature Communications. 10 (1), 1930(2019).</li>
<li>Rodriguez, D. L., Quail, M. M., Hernday, A. D., Nobile, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+circuits+regulating+developmental+processes+in+Candida+albicans%5BTitle%5D)+AND+%22Frontiers+in+Cellular+and+Infection+Microbiology%22%5BJournal%5D)">Transcriptional circuits regulating developmental processes in Candida albicans.</a> Frontiers in Cellular and Infection Microbiology. 10, 605711(2020).</li>
<li>Zhu, Q., Liu, N., Orkin, S. H., Yuan, G. -C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CUT%26amp%3BRUNTools%3A+a+flexible+pipeline+for+CUT%26amp%3BRUN+processing+and+footprint+analysis%5BTitle%5D)+AND+%22Genome+Biology%22%5BJournal%5D)">CUT&amp;RUNTools: a flexible pipeline for CUT&amp;RUN processing and footprint analysis.</a> Genome Biology. 20 (1), 192(2019).</li>
<li>Teytelman, L., Thurtle, D. M., Rine, J., Van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+expressed+loci+are+vulnerable+to+misleading+ChIP+localization+of+multiple+unrelated+proteins%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (46), 18602-18607 (2013).</li>
</ol>

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

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&amp;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 ...

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-specific manner4. Recently, chromatin regulators and histone modifiers have also emerged as important regulators of C. albicans biofilm formation5 and morphogenesis6 by mediating DNA accessibility. To understand the complex biology of this important fungal pathogen, effective methods to determine the genome-wide localization of specific TFs during distinct developmental and cellular processes is valuable.
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a widely used method to investigate protein-DNA interactions in C. albicans5,6 and has largely replaced the more classical chromatin immunoprecipitation followed by microarray (ChIP-chip)9 method. Both ChIP-seq and ChIP-chip methods, however, require a large number of input cells10, which can be a complicating factor when investigating TFs in the context of specific samples and modes of growth, such as biofilms collected from patients or animal models of infection. In addition, the chromatin immunoprecipitation (ChIP) assay often yields a significant amount of background signal throughout the genome, requiring a high level of enrichment for the target of interest to sufficiently separate signal from noise. While the ChIP-chip assay is largely outdated today, the sequencing depths necessary for ChIP-seq make this assay prohibitively expensive for many researchers, particularly those studying multiple TFs and/or chromatin-associated proteins.

Cleavage under targets and release using nuclease (CUT&#38;RUN) is an attractive alternative to ChIP-seq. It was developed by the Henikoff lab in 2017 to circumvent the limitations of ChIP-seq and chromatin endogenous cleavage followed by sequencing ChEC-seq11,12, another method to identify protein-DNA interactions on a genome-wide level, while providing high-resolution, genome-wide mapping of TFs and chromatin-associated proteins13. CUT&#38;RUN relies on the targeted digestion of chromatin within permeabilized nuclei using tethered micrococcal nucleases, followed by sequencing of the digested DNA fragments9,10. As DNA fragments are specifically generated at the loci that are bound by a protein of interest, rather than being generated throughout the genome via random fragmentation as in ChIP assays, the CUT&#38;RUN approach results in greatly reduced background signals and, thus, requires 1/10th&#160;of the sequencing depth compared to ChIP-seq11,13,14. These improvements ultimately lead to significant reductions in sequencing costs and reductions in the total number of input cells needed as starting material for each sample.
Here, a robust CUT&#38;RUN protocol is described that has been adapted and optimized for determining the genome-wide localization of TFs in C. albicans cells isolated from biofilms and planktonic cultures. A thorough data analysis pipeline is also presented, which enables the processing and analysis of the resulting sequence data and requires users to have minimal expertise in coding or bioinformatics. Briefly, this protocol describes epitope tagging of TF-coding genes, harvesting of biofilm and planktonic cells, isolation of intact permeabilized nuclei, incubation with primary antibodies against the specific protein or epitope-tagged protein of interest, tethering of the chimeric A/G-micrococcal nuclease (pAG-MNase) fusion proteins to the primary antibodies, genomic DNA recovery after chromatin digestion, and preparation of genomic DNA libraries for sequencing.
The experimental CUT&#38;RUN protocol is followed by a purpose-built data analysis pipeline, which takes raw DNA sequencing reads in FASTQ format and implements all required processing steps to provide a complete list of significantly enriched loci bound by the TF of interest (targeted by the primary antibody). Note that multiple steps of the described library preparation protocol have been specifically adapted and optimized for CUT&#38;RUN analysis of TFs (as opposed to nucleosomes). While the data presented in this manuscript were generated using TF-specific adaptations of a commercial CUT&#38;RUN kit, these protocols have also been validated using individually sourced components (i.e., pAG-MNase enzyme and magnetic DNA purification beads) and in-house-prepared buffers, which can significantly reduce experimental cost. The comprehensive experimental and data analysis protocols are described in detail below in a step-by-step format. All reagents and critical equipment, as well as buffer and media recipes, are listed in the Table of Materials and Supplementary File 1, respectively.

<table><tbody><tr><td>0.22 &amp;mu;m filter</td><td>Millipore Sigma</td><td>SLGPM33RS</td><td></td></tr><tr><td>0.65 mL low-adhesion tubes</td><td>VWR</td><td>490003-190</td><td></td></tr><tr><td>1 M CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific</td><td>50-152-341</td><td></td></tr><tr><td>1 M PIPES</td><td>Fisher Scientific</td><td>AAJ61224AK</td><td></td></tr><tr><td>12-well untreated cell culture plates</td><td>Corning</td><td>351143</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>60-24-2</td><td></td></tr><tr><td>2% Digitonin</td><td>Fisher Scientific</td><td>CHR103MI</td><td></td></tr><tr><td>50 mL conical tubes</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>5x phusion HF buffer</td><td>Fisher Scientific</td><td>F530S</td><td>Item part of the Phusion high fidelity DNA polymerase; referred to in text as &quot;DNA polymerase buffer&quot;</td></tr><tr><td>Agar</td><td>Criterion</td><td>C5001</td><td></td></tr><tr><td>Agencourt AMPure XP magnetic beads</td><td>Beckman Coulter</td><td>A63880</td><td></td></tr><tr><td>Agilent Bioanalyzer</td><td>Agilent</td><td>G2939BA</td><td>Referred to in the text as &quot;capillary electrophoresis instrument&quot;; user-dependepent</td></tr><tr><td>Amplitube PCR reaction strips with attached caps, Simport Scientific</td><td>VWR</td><td>89133-910</td><td></td></tr><tr><td>Bacto peptone</td><td>BD Biosciences</td><td>211677</td><td></td></tr><tr><td>Benchling primer design tool</td><td>Benchling</td><td></td><td>https://www.benchling.com/molecular-biology/; Referred to in the text as &quot;the primer design tool&quot;</td></tr><tr><td>Betaine</td><td>Fisher Scientific</td><td>AAJ77507AB</td><td></td></tr><tr><td>Calcofluor white stain</td><td>Sigma-Aldrich</td><td>18909-100ML-F</td><td></td></tr><tr><td>&lt;em&gt;Candida &lt;/em&gt;Genome Database</td><td></td><td></td><td>http://www.candidagenome.org/</td></tr><tr><td>Concanavalin A (ConA) conjugated paramagnetic beads</td><td>Polysciences</td><td>&amp;nbsp;86057-3</td><td></td></tr><tr><td>Conda software</td><td></td><td></td><td>https://docs.conda.io/en/latest/miniconda.html</td></tr><tr><td>curl tool</td><td></td><td></td><td>http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly21/current/C_albicans_SC5314_A21_current_&lt;br/&gt; chromosomes.fasta.gz</td></tr><tr><td>CUTANA ChIC/CUT&amp;amp;RUN kit</td><td>Epicypher</td><td>14-1048</td><td>Referred to in the text as &quot;the CUT&amp;amp;RUN kit&quot;</td></tr><tr><td>Deoxynucleotide (dNTP) solution mix (10 mM)</td><td>New England Biolabs</td><td>N0447S</td><td></td></tr><tr><td>Dextrose (D-glucose)</td><td>Fisher Scientific</td><td>D163</td><td></td></tr><tr><td>Difco D-mannitol</td><td>&amp;nbsp;BD Biosciences</td><td>217020</td><td></td></tr><tr><td>Disposable cuvettes</td><td>Fisher Scientific</td><td>14-955-127</td><td></td></tr><tr><td>Disposable transfer pipets</td><td>Fisher Scientific</td><td>13-711-20</td><td></td></tr><tr><td>DNA Gel Loading Dye (6x)</td><td>Fisher Scientific</td><td>R0611</td><td></td></tr><tr><td>DreamTaq green DNA polymerase</td><td>Fisher Scientific</td><td>EP0713</td><td>Referred to in the text as &quot;cPCR DNA polymerase&quot;</td></tr><tr><td>DreamTaq green DNA polymerase buffer</td><td>Fisher Scientific</td><td>EP0713</td><td>Item part of the DreamTaq green DNA polymerase; referred to in the text as &quot;cPCR DNA polymerase buffer&quot;</td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;spike-in DNA</td><td>Epicypher</td><td>18-1401</td><td></td></tr><tr><td>ELMI Microplate incubator</td><td>ELMI</td><td>TRMS-04</td><td>Referred to in the text as &quot;microplate incubator&quot;</td></tr><tr><td>End Prep Enzyme Mix</td><td></td><td></td><td>Item part of the NEBNext Ultra II DNA Library Prep kit</td></tr><tr><td>End Prep Reaction Buffer</td><td></td><td></td><td>Item part of the NEBNext Ultra II DNA Library Prep kit</td></tr><tr><td>Ethanol 200 proof</td><td>VWR</td><td>89125-170</td><td></td></tr><tr><td>FastDigest MssI</td><td>Fisher Scientific</td><td>FD1344</td><td>Referred to in the text as &quot;restriction enzyme&quot;</td></tr><tr><td>FastDigest MssI Buffer</td><td>Fisher Scientific</td><td>FD1344</td><td>Item part of the FastDigest MssI kit; referred to in the text as &quot;restriction enzyme buffer&quot;</td></tr><tr><td>Ficoll 400</td><td>Fisher BioReagents</td><td>BP525-25</td><td></td></tr><tr><td>Fluorescence microscope</td><td></td><td></td><td>User-dependent</td></tr><tr><td>Gel electrophoresis apparatus</td><td></td><td></td><td>User-dependent</td></tr><tr><td>GeneRuler low range DNA ladder</td><td>Fisher Scientific</td><td>FERSM1192</td><td></td></tr><tr><td>GitBash workflow</td><td></td><td></td><td>https://gitforwindows.org/</td></tr><tr><td>GitHub source code</td><td></td><td></td><td>https://github.com/akshayparopkari/cut_run_analysis</td></tr><tr><td>HEPES-KOH pH 7.5</td><td>Boston BioProducts</td><td>BBH-75-K</td><td></td></tr><tr><td>High-speed centrifuge</td><td></td><td></td><td>User-dependent</td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>PX1830-4</td><td></td></tr><tr><td>Lens paper</td><td>VWR</td><td>52846-001</td><td></td></tr><tr><td>Ligation Enhancer</td><td></td><td></td><td>Item part of the NEBNext Ultra II DNA Library Prep kit</td></tr><tr><td>Lithium acetate dihydrate</td><td>MP Biomedicals</td><td>215525683</td><td></td></tr><tr><td>Living Colors Full-Length GFP polyclonal antibody</td><td>Takara</td><td>632592</td><td>User-dependent</td></tr><tr><td>MACS2</td><td></td><td></td><td>https://pypi.org/project/MACS2/</td></tr><tr><td>Magnetic separation rack, 0.2 mL tubes</td><td>Epicypher</td><td>10-0008</td><td></td></tr><tr><td>Magnetic separation rack, 1.5 mL tubes</td><td>Fisher Scientific</td><td>MR02</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5 mL</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Microplate and cuvette spectrophotometer</td><td>BioTek</td><td>EPOCH2TC</td><td>Referred to in the text as &quot;spectrophotometer&quot;; user-dependent</td></tr><tr><td>MochiView</td><td></td><td></td><td>http://www.johnsonlab.ucsf.edu/mochiview-downloads</td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>M3183</td><td></td></tr><tr><td>NaCl</td><td>VWR</td><td>470302-522</td><td></td></tr><tr><td>NaOH</td><td>Fisher Scientific</td><td>S318-500</td><td></td></tr><tr><td>NCBI GEO</td><td></td><td></td><td>https://www.ncbi.nlm.nih.gov/geo/</td></tr><tr><td>NEBNext Adaptor for Illumina</td><td></td><td></td><td>Item part of the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1); referred to in the text as &quot;Adapter&quot;</td></tr><tr><td>NEBNext Index X Primer for Illumina</td><td></td><td></td><td>Item part of the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1); referred to in the text as &quot;Reverse Uniquely Indexed Library Amplification Primer&quot;</td></tr><tr><td>NEBNext Multiplex Oligos for Illumina (Index Primers Set 1)</td><td>New England Biolabs</td><td>E7335S</td><td></td></tr><tr><td>NEBNext Ultra II DNA Library Prep kit</td><td>New England Biolabs</td><td>E7645S</td><td>Referred to in the text as &quot;library prep kit&quot;</td></tr><tr><td>NEBNext Universal PCR Primer for Illumina</td><td></td><td></td><td>Item part of the NEBNext Multiplex Oligos for Illumina (Index Primers Set 1); referred to in the text as &quot;Universal Forward Library Amplification Primer&quot;</td></tr><tr><td>Nourseothricin sulfate (NAT)</td><td>Goldbio</td><td>N-500-2</td><td></td></tr><tr><td>Novex TBE Gels, 10%, 15 well</td><td>Fisher Scientific</td><td>EC62755BOX</td><td></td></tr><tr><td>Nutating mixer</td><td>VWR</td><td>82007-202</td><td></td></tr><tr><td>Nutrient broth</td><td>Criterion</td><td>C6471</td><td></td></tr><tr><td>pADH110</td><td>Addgene</td><td>90982</td><td>Referred to in the text as &quot;plasmid repository ID# 90982&quot;</td></tr><tr><td>pADH119</td><td>Addgene</td><td>90985</td><td>Referred to in the text as &quot;plasmid repository ID# 90985&quot;</td></tr><tr><td>pADH137</td><td>Addgene</td><td>90986</td><td>Referred to in the text as &quot;plasmid repository ID# 90986&quot;</td></tr><tr><td>pADH139</td><td>Addgene</td><td>90987</td><td>Referred to in the text as &quot;plasmid repository ID# 90987&quot;</td></tr><tr><td>pADH140</td><td>Addgene</td><td>90988</td><td>Referred to in the text as &quot;plasmid repository ID# 90988&quot;</td></tr><tr><td>pAG-MNase</td><td>Epicypher</td><td>15-1016 or 15-1116</td><td>50 rxn or 250 rxn</td></tr><tr><td>pCE1</td><td>Addgene</td><td>174434</td><td>Referred to in the text as &quot;plasmid repository ID# 174434&quot;</td></tr><tr><td>Petri dishes with clear lid</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Phusion high fidelity DNA polymerase</td><td>Fisher Scientific</td><td>F530S</td><td>Referred to in the text as &quot;DNA polymerase&quot;</td></tr><tr><td>Polyethylene glycol (PEG) 3350</td><td>VWR</td><td>10791-816</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Fisher Scientific</td><td>P285-500</td><td></td></tr><tr><td>Qubit 1x dsDNA HS assay kit</td><td>Invitrogen</td><td>Q33230</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Life Technologies</td><td>Q33216</td><td>Referred to in the text as &quot;fluorometer&quot;; user-dependent</td></tr><tr><td>Rabbit IgG negative control antibody</td><td>Epicypher</td><td>13-0042</td><td></td></tr><tr><td>RNase A</td><td>Sigma-Aldrich</td><td>10109169001</td><td></td></tr><tr><td>Roche Complete protease inhibitor (EDTA-free) tablets</td><td>Sigma-Aldrich</td><td>5056489001</td><td></td></tr><tr><td>RPMI-1640</td><td>Sigma-Aldrich</td><td>R6504</td><td></td></tr><tr><td>Shaking incubator</td><td>Eppendorf</td><td>M12820004</td><td>User-dependent</td></tr><tr><td>Sorbitol</td><td>Sigma-Aldrich</td><td>S1876-500G</td><td></td></tr><tr><td>Spin-X centrifuge tube filters</td><td>Fisher Scientific</td><td>07-200-385</td><td></td></tr><tr><td>Sterile inoculating loops</td><td>VWR</td><td>30002-094</td><td></td></tr><tr><td>SYBR Gold nucleic acid gel stain</td><td>Fisher Scientific</td><td>S11494</td><td></td></tr><tr><td>SYTO 13 nucleic acid stain</td><td>Fisher Scientific</td><td>S7575</td><td>Referred to in the text as &quot;nucleic acid gel stain&quot;</td></tr><tr><td>Thermocycler</td><td></td><td></td><td>User-dependent</td></tr><tr><td>ThermoMixer C</td><td>Eppendorf</td><td>5382000023</td><td></td></tr><tr><td>Tris (hydroxymethyl) aminomethane</td><td>Sigma-Aldrich</td><td>252859-100G</td><td></td></tr><tr><td>Ultra II Ligation Master Mix</td><td></td><td></td><td>Item part of the NEBNext Ultra II DNA Library Prep kit; referred to in the text as &quot;Ligation Master Mix&quot;</td></tr><tr><td>Ultra II Q5 Master Mix</td><td></td><td></td><td>Item part of the NEBNext Ultra II DNA Library Prep kit; referred to in the text as &quot;High Fidelity DNA Polymerase Master Mix &quot;</td></tr><tr><td>UltraPure salmon sperm DNA solution</td><td>Invitrogen</td><td>15632011</td><td></td></tr><tr><td>USER Enzyme</td><td></td><td></td><td>Item part of the NEBNext Ultra II DNA Library Prep kit; referred to in the text as &quot;Uracil Excision Enzyme&quot;</td></tr><tr><td>Vortex mixer</td><td>VWR</td><td>10153-834</td><td></td></tr><tr><td>wget tool</td><td></td><td></td><td>http://www.candidagenome.org/download/sequence/C_albicans_SC5314/Assembly21/current/C_albicans_SC5314_A21_current_&lt;br/&gt; chromosomes.fasta.gz</td></tr><tr><td>Yeast extract</td><td>Criterion</td><td>C7341</td><td></td></tr><tr><td>Zymolyase 100T (lyticase, yeast lytic enzyme)</td><td>Fisher Scientific</td><td>NC0439194</td><td></td></tr></tbody></table>

genome-wide profiling of transcription factor-dna binding interactions in candida albicans: a comprehensive cut&amp;run method and data analysis workflow

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

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&amp;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&amp;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&amp;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&amp;RUN data analysis.

This robust CUT&amp;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&amp;RUN sequencing data and requires users to have minimal expertise in coding or bioinformatics (see Figure 3 for an overview of t...

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

Regulatory transcription factors control many important biological processes, including cellular differentiation, responses to environmental perturbations ...

genome-wide-profiling-transcription-factor-dna-binding-interactions

Research

JoVE Journal

Biology

We developed a protocol that is fast, sensitive and reproducible for pathogen gene expression profiling during an infection.

Gene Expression Profiling of Infecting Microbes Using a Digital Bar-coding Platform

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Here we present a novel method for determining binding affinities at equilibrium and in solution with high sensitivity on a large scale. This improves the quantitative analysis of transcription factor-DNA binding. The method is based on automated fluorescence anisotropy measurements in a controlled delivery system.

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &gt;1,000 TFs in a single experiment.

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

The protocol presented here describes a complete pipeline to analyze RNA-sequencing transcriptome data from raw reads to functional analysis, including quality control and preprocessing steps to advanced statistical analytical approaches.

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

CUT&amp;RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&amp;RUN.

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

An optimized Cleavage Under Targets and Release Using Nuclease followed by next generation sequencing (CUT&amp;RUN-seq) protocol is described for neuroendocrine small cell lung cancer cell lines. It enables genome-wide mapping of various histone modifications and transcription factor (e.g. E2F7) binding sites to investigate epigenetic and transcriptional deregulation in SCLC pathobiology.

Genome-Wide Mapping of Histone Modifications and Transcription Factor Binding Sites in Neuroendocrine Small Cell Lung Cancer Cell Lines Using CUT&amp;RUN

Here, we present a quick and simple ATAC-seq protocol to be implemented in L4-stage Caenorhabditis elegans whole-worm using only 30 µL of worm pellet.

Chromatin Accessibility Profiling in Whole Caenorhabditis elegans L4 Larvae

Learn CUT&RUN for genome-wide profiling of transcription factor-DNA binding in Candida albicans. This computational workflow offers faster, more sensitive analysis than traditional ChIP methods for pathogen research.

Genome-wide Profiling of Transcription Factor-DNA Binding Interactions in Candida albicans: A Comprehensive CUT and RUN Method and Data Analysis Workflow (Video) | JoVE Journal

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

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