detecting protein-dna interactions using cut&amp;run analysis

Beginner-Friendly Single-Language Pipeline for CUT&RUN Data Analysis

The ability to measure interactions between proteins and genomic DNA is fundamental to understanding the biology of chromatin regulation. Effective assays that measure chromatin occupancy for a given protein provide at least two key pieces of information: i) genomic localization and ii) protein abundance at a given genomic region. Tracking the recruitment and localization changes of a protein of interest in chromatin can reveal direct target loci of the protein and reveal mechanistic roles of that protein in chromatin-based biological processes such as regulation of transcription, DNA repair, or DNA replication. The techniques available today to profile protein-DNA interactions are enabling researchers to explore regulation at unprecedented resolution. Such technical advances have been enabled through the introduction of new chromatin profiling techniques that include the development of Cleavage Under Targets and Release Using Nuclease (CUT&#38;RUN) by the Henikoff laboratory. CUT&#38;RUN offers several technical advantages over conventional chromatin immunoprecipitation (ChIP) that include lower cell input requirements, lower sequencing depth requirements, and increased sensitivity with reduced background signal due to a lack of cross-linking agents that otherwise mask antibody epitopes. Adopting this technique to study chromatin regulation requires a thorough understanding of the principle underlying the technique, and an understanding of how to analyze, validate, and interpret CUT&#38;RUN data.
The CUT&#38;RUN procedure begins with binding of cells to Concanavalin A conjugated to magnetic beads to enable manipulation of low cell numbers throughout the procedure. Isolated cells are permeabilized using a mild detergent to facilitate introduction of an antibody that targets the protein of interest. Micrococcal nuclease (MNase) is then recruited to the bound antibody using a Protein A or Protein A/G tag tethered to the enzyme. Calcium is introduced to initiate enzymatic activity. MNase digestion results in mono-nucleosomal DNA-protein complexes. Calcium is subsequently chelated to end the digestion reaction, and short DNA fragments from the MNase digestion are released from nuclei, then subjected to DNA purification, library preparation, and high-throughput sequencing1 (Figure 1).
In silico approaches to map and quantify protein occupancy across the genome have developed in parallel with the wet lab approaches used to enrich those DNA-protein interactions. Identification of regions of enriched signals (peaks) is one of the most critical steps in the bioinformatics analysis. Initial ChIP-seq analysis methods used algorithms such as MACS2 and SICER3, which employed statistical models to distinguish bona fide protein-DNA binding sites from background noise. However, the lower background noise and higher resolution of CUT&#38;RUN data render some peak calling programs employed in ChIP-seq analysis unsuitable for CUT&#38;RUN analysis4. This challenge highlights the need for new tools better suited for the analysis of CUT&#38;RUN data. SEACR4 represents one such tool recently developed to enable peak calling from CUT&#38;RUN data while overcoming limitations associated with tools typically employed toward ChIP-seq analysis.
Biological interpretations from CUT&#38;RUN sequencing data are drawn from the outputs downstream of peak calling in the analysis pipeline. Several functional annotation programs can be implemented to predict the potential biological relevance of the called peaks from CUT&#38;RUN data. For example, the Gene Ontology (GO) project provides well-established functional identification of genes of interest5,6,7. Various software tools and resources facilitate GO analysis to reveal genes and gene sets enriched amongst CUT&#38;RUN peaks8,9,10,11,12,13,14. Furthermore, visualization software such as Deeptools15, Integrative genomics viewer (IGV)16, and UCSC Genome Browser17 enable visualization of signal distribution and patterns at regions of interest across the genome.
The ability to draw biological interpretations from CUT&#38;RUN data depends critically upon validation of data quality. Critical components to validate include the assessment of: i) CUT&#38;RUN library sequencing quality, ii) replicate similarity, and iii) signal distribution at peak centers. Completing the validation of all three components is crucial to ensure the reliability of CUT&#38;RUN library samples and downstream analysis results. Therefore, it is essential to establish introductory CUT&#38;RUN analysis guides to enable bioinformatics beginners and wet lab researchers to conduct such validation steps as part of their standard CUT&#38;RUN analysis pipelines.
Alongside the development of wet lab CUT&#38;RUN experiment, various in silico CUT&#38;RUN analysis pipelines, such as CUT&#38;RUNTools 2.018,19, nf-core/cutandrun20, and CnRAP21, have been developed to support CUT&#38;RUN data analysis. These tools provide powerful approaches to analyzing single-cell and bulk CUT&#38;RUN and CUT&#38;Tag datasets. However, the relatively complex modular program structure and required familiarity with multiple programming languages to conduct these analysis pipelines may hinder adoption by bioinformatics beginners who seek to thoroughly understand the CUT&#38;RUN analysis steps and customize their own pipelines. Circumvention of this barrier requires a new introductory CUT&#38;RUN analysis pipeline that is provided in simple step-by-step scripts encoded using a simple single programming language.
In this article, we describe a simple single-language CUT&#38;RUN analysis pipeline protocol that provides step-by-step scripts supported with detailed descriptions to enable new and novice users to conduct CUT&#38;RUN sequencing analysis. Programs used in this pipeline are publicly available by the original developer groups. Major steps described in this protocol include read alignment, peak calling, functional analysis and, most critically, validation steps to assess sample quality to determine data suitability and reliability for biological interpretation (Figure 2). Furthermore, this pipeline provides users the opportunity to cross-reference analysis results against publicly available CUT&#38;RUN datasets. Ultimately, this CUT&#38;RUN analysis pipeline protocol serves as an introductory guide and reference for bioinformatic analysis beginners and wet lab researchers.

Author Spotlight: Investigating the Role of Repetitive DNA Misregulation in Cancer Initiation and Immunotherapy Resistance

Introductory Analysis and Validation of CUT&#38;RUN Sequencing Data

Our research explores the role of repetitive DNA elements in cancer initiation and progression. We ask whether these elements are directly involved in mechanisms of cancer initiation, and, if so, can we use this information to improve patient outcomes. Many next-generation sequencing technology and analytic pipeline has been developed to capture the feature and changes of the epigenetic factors such as transcriptional or the post-translational chromatin changes and the binding at the specific localization on the chromatin and sequences of DNA or RNA which are captured by the defensive protein in both bulk and single-cell resolution.The technologies used to advance research in our field are those that can map the genomic positions of modified histones or transcription factors on chromatin. For our purposes, the most relevant technique within this umbrella of technologies is a technique called CUT&RUN. Many publicly available CUT&RUN analysis pipeline are relatively complex for the bioinformatic beginner who want to understand the flow of the analysis and to develop their own analysis pipeline for their project.But our CUT&RUN analysis pipeline are developed in step-by-step manner in single program language, which are much easier to understand the flow and easier to modify to get their publication-quality data and to build up the experience for the bioinformatics. We are key focusing on to find the answer about how chronic double-stranded RNA expression affect the biomimicry evasion and the immunotherapy rates in cancer development, and how TP53 protein can regulate the chronic double-stranded RNA expression and the biomimicry evasion. And at the end, we want to understand how we can cure the immunotherapy resistant cancer by understanding and targeting the chronic double-stranded RNA expression and the biomimicry evasion process.

Detecting Protein-DNA Interactions Using CUT&amp;RUN Analysis

To begin, download the compressed analysis pipeline from GitHub by typing the command in the terminal. After downloading, enter the command to decompress the zip file. Once the decompression is complete, remove the zip file and change the decompressed folder name.Then, set executable permissions for all the shell scripts in the working directory. From the available options, locate the appropriate installation shell script. Look for a script named Script_01_Installation.sh.Open the terminal and type echo SHELL to check the default shell used in the active terminal. If bash is not the default shell, set it by typing chsh s in the terminal. Type the fast queue download script in the terminal, or drag the script file into the terminal and press enter.After running the script, check the log file for error messages. Now, enter the trimming script in the terminal and execute it. Next, type the bowtie2 index creation script in the terminal and execute it.Finally, run the bowtie2 mapping script and press enter. Type the bam filtering and sorting script in the terminal and execute it. Next, convert bam to BEDPE, BED, and bedGraph formats by typing the appropriate command in the terminal and pressing enter.Perform normalization using the SRPMC method, then run the insert size analysis script. Perform peak calling using the MACS command in the terminal. Afterward, type the command for peak calling using SEACR and press enter.Create peak BED files by using the appropriate command in the terminal, and then filter the peaks. Next, enter the command to merge MACS peaks. Type the command in the terminal to merge SEACR peaks.Generate a correlation plot, and run the command for principle component analysis in the terminal. Then, create a Venn diagram comparing different methods using the appropriate command. Enter the command to generate a Venn diagram for replicates.Finally, generate a focused peak center heat map. Then, generate a whole peak center heat map.

detecting-protein-dna-interactions-using-cutrun-analysis

Research

JoVE Journal

Genetics

102 visualizações. Para começar, baixe o pipeline de análise compactado do GitHub digitando o comando no terminal. Após o download, digite o comando para descompactar o arquivo zip. Quando a descompactação estiver concluída, remova o arquivo zip e altere o nome da pasta descompactada.Em seguida, defina permissões executáveis para todos os scripts de shell no diretório de trabalho. Nas opções disponíveis, localize o script de shell de instalação apropriado. ...