Systems Biology

Protocols in Current Issue
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0 Q&A 2323 Views Jun 20, 2022

Chromatin accessibility is a key determinant of gene expression that can be altered under different physiological and disease conditions. Skeletal muscle is made up of myofibers that are highly plastic and adaptive. Therefore, assessing the genome-wide chromatin state of myofibers under various conditions is very important to gain insight into the epigenetic state of myonuclei. The rigid nature of myofibers, as well as the low number of myonuclei that they contain, have rendered genome-wide studies with myofibers challenging. In recent years, ATAC-Seq from whole muscle and single nucleus ATAC-Seq have been performed. However, these techniques cannot distinguish between different fiber and cell types present in the muscle. In addition, due to the limited depth capacity obtained from single nucleus ATAC-Seq, an extensive comparative analysis cannot be performed. Here, we introduce a protocol where we combine the isolation of a single myofiber with OMNI ATAC-Seq. This protocol allows for genome-wide analysis of accessible chromatin regions of a selected single myofiber at a sufficient depth for comparative analysis under various physiological and disease conditions. This protocol can also allow for a specific myofiber to be selected, such as a regenerating myofiber. In the future, this protocol can help identify global changes in chromatin state under various conditions, as well as between different types of myofibers.

Graphical abstract:

1 Q&A 1594 Views May 5, 2022

DNA methylation is a conserved chemical modification, by which methyl groups are added to the cytosine of DNA molecules. Methylation can influence gene expression without changing the sequence of a particular gene. This epigenetic effect is an intriguing phenomenon that has puzzled biologists for years. By probing the temporal and spatial patterns of DNA methylation in genomes, it is possible to learn about the biological role of cytosine methylation, as well as its involvement in gene regulation and transposon silencing. Advances in whole-genome sequencing have led to the widespread adoption of methods that examine genome-wide patterns of DNA methylation. Achieving sufficient sequencing depth in these types of experiments is costly, particularly for pilot studies in organisms with large genome sizes, or incomplete reference genomes. To overcome this issue, assays to determine site-specific DNA methylation can be used. Although often used, these assays are rarely described in detail. Here, we describe a pipeline that applies traditional TA cloning, Sanger sequencing, and online tools to examine DNA methylation. We provide an example of how to use this protocol to examine the pattern of DNA methylation at a specific transposable element in maize.

0 Q&A 5099 Views Mar 5, 2022

In recent years, DNA methylation research has been accelerated by the advent of nanopore sequencers. However, read length has been limited by the constraints of base conversion using the bisulfite method, making analysis of chromatin content difficult. The read length of the previous method combining bisulfite conversion and long-read sequencing was ~1.5 kb, even using targeted PCR. In this study, we have improved read length (~5 kb), by converting unmethylated cytosines to uracils with APOBEC enzymes, to reduce DNA fragmentation. The converted DNA was then sequenced using a PromethION nanopore sequencer. We have also developed a new analysis pipeline that accounts for base conversions, which are not present in conventional nanopore sequencing, as well as errors produced by nanopore sequencing.

1 Q&A 2672 Views Nov 5, 2021

Characterizing the molecular mechanisms regulating gene expression is crucial for understanding the regulatory processes underlying physiological responses to environmental and developmental signals in eukaryotes. The covalent modification of histones contributes to the compaction levels of chromatin, as well as the recruitment of the transcriptional machinery to specific loci, facilitating metastable changes in gene activity. ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) has become the gold standard method for determining histone modification profiles among different organisms, tissues, and genotypes. In the current protocol, we describe a highly robust method for performing ChIP-seq of histone modifications in Arabidopsis thaliana plantlets. Besides its robustness, this method uses in-house-prepared buffers for chromatin extraction, immunoprecipitation, washing, and elusion, making it cost-effective in contrast to commercial kits.

0 Q&A 1658 Views Sep 20, 2021

In the field of chromatin biology, a major goal of understanding the roles of histone post-translational modifications is to identify the proteins and domains that recognize these modifications. Synthetic histone peptides containing one or more modifications are a key tool to probe these interactions in pull-down assays with recombinant proteins or cell lysates. Building on these approaches, the binding specificity of a protein of interest can be screened against many histone peptides in parallel using a peptide array. In this protocol, we describe the expression and purification of a recombinant protein of interest in bacteria, followed by an assay for binding to histone post-translational modifications using a commercially available histone peptide array. The purification uses a versatile dual-tagging and cleavage strategy and equipment commonly available in a molecular biology laboratory.

Graphic abstract:

Overview of protocol for purifying recombinant protein and hybridizing to a histone peptide array.

0 Q&A 2711 Views Aug 5, 2021

Primary somatosensory neurons, whose cell bodies reside in the dorsal root ganglion (DRG) and trigeminal ganglion, are specialized to transmit sensory information from the periphery to the central nervous system. Our molecular understanding of peripheral sensory neurons has been limited by both their heterogeneity and low abundance compared with non-neuronal cell types in sensory ganglia. We describe a protocol to isolate nuclei from mouse DRGs using iodixanol density gradient centrifugation, which enriches for neuronal nuclei while still sampling non-neuronal cells such as satellite glia and Schwann cells. This protocol is compatible with a range of downstream applications such as single-nucleus transcriptional and epigenomic assays.

0 Q&A 4230 Views Jun 5, 2021

We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells. With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg++ activates the transposase and inserts sequencing adapters into adjacent DNA in situ. We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying the transposase activation conditions when using histone H3K4me2, H3K4me3, or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, we redirect the tagmentation of accessible DNA sites to produce chromatin accessibility maps with exceptionally high signal-to-noise and resolution. All steps from nuclei to amplified sequencing-ready libraries are performed in single PCR tubes using non-toxic reagents and inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench, or classroom.

0 Q&A 2567 Views Jun 5, 2021

DNA methylation in gene promoters plays a major role in gene expression regulation, and alterations in methylation patterns have been associated with several diseases. In this context, different software suites and statistical methods have been proposed to analyze differentially methylated positions and regions. Among them, the novel statistical method implemented in the mCSEA R package proposed a new framework to detect subtle, but consistent, methylation differences. Here, we provide an easy-to-use pipeline covering all the necessary steps to detect differentially methylated promoters with mCSEA from Illumina 450K and EPIC methylation BeadChips data. This protocol covers the download of data from public repositories, quality control, data filtering and normalization, estimation of cell type proportions, and statistical analysis. In addition, we show the procedure to compare disease vs. normal phenotypes, obtaining differentially methylated regions including promoters or CpG Islands. The entire protocol is based on R programming language, which can be used in any operating system and does not require advanced programming skills.

0 Q&A 19624 Views Aug 20, 2018
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a routine procedure in the lab; however, epigenome-wide quantitative comparison among independent ChIP-seq experiments remains a challenge. Here, we contribute an experimental protocol combined with a computational workflow allowing quantitative and comparative assessment of epigenome using animal tissues.
0 Q&A 7921 Views Aug 5, 2018
Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was measured by amplification of selected ligation junctions corresponding to a small number of genomic loci (‘one versus one’) through semi-quantitative PCR (Dekker et al., 2002). The chromosome conformation capture-on-chip (4C) and chromosome conformation capture carbon copy (5C) technologies then extended 3C to count ligation events in a ‘one versus many’ or ‘many versus many’ manner, respectively. Hi-C (Lieberman-Aiden et al., 2009) finally combined 3C with next-generation sequencing (Metzker, 2010). Here, before religation sticky ends are filled in with biotin-labeled nucleotide analogs to enrich for fragments with a ligation junction in a later step. The Hi-C libraries are then subjected to high-throughput sequencing and the resultant reads mapped to a reference genome, allowing the determination of contact probabilities in a ‘many versus many’ way with a resolution that is limited only by the distribution of restriction sites and the read depth. The first application of Hi-C was the elucidation of global chromatin folding principles in the human genome (Lieberman-Aiden et al., 2009). Similar efforts have since been carried out in other eukaryotic model species such as yeast (Duan et al., 2010), Drosophila (Sexton et al., 2012) and Arabidopsis (Grob et al., 2014; Wang et al., 2015; Liu et al., 2016). Other uses of Hi-C include the study of chromatin looping at high-resolution (Rao et al., 2014; Liu et al., 2016), of chromatin reorganization along the cell cycle (Naumova et al., 2013) and of differences in chromatin organization in mutant individuals (Feng et al., 2014). The tethered conformation capture protocol (TCC) (Kalhor et al., 2011) described here is a variant of the original Hi-C method (Lieberman-Aiden et al., 2009) and was adapted to Triticeae.

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