Name: atac-seq assay with low mitochondrial dna contamination from primary human cd4+ t lymphocytes
Uploaded: 2019-03-22T14:00:00
Description: Watch this Scientific Journal Video about ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes at JoVE.com

We thank Atsede Siba for technical support. C.S.C. is supported by NIH grant 1R61DA047032.

This improved protocol provides step-by-step instructions for performing ATAC-seq of CD4+ lymphocytes, from the starting material of whole blood through data analysis (Figure 1).
1. Isolation of CD4+ T Cells from Whole Blood
NOTE: The starting material for this protocol is 15 mL of fresh whole blood collected using standard procedures, allowing the source of the starting material to be selected based on research requireme...

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The modified ATAC-seq protocol presented in this article provides reproducible results with minimal mitochondrial DNA contamination. The protocol has been used to successfully characterize chromatin architecture of human primary PBMCs10, human monocytes, mouse dendritic cells (unpublished), and cultured melanoma cell lines11. We anticipate this improved lysis condition has the potential to work for other cell types as well. It is also anticipated that this nuclei isolation ...

The assay for transposase-accessible chromatin sequencing (ATAC-seq) has rapidly become the leading method for interrogating chromatin architecture. ATAC-seq can identify regions of accessible chromatin through the process of tagmentation, the fragmenting and tagging of DNA by the same enzyme, to produce libraries with which sequencing can determine chromatin accessibility across an entire genome. This tagmentation process is mediated by the hyperactive Tn5 transposase, which only cuts open regions of chromatin due to nucleosomic steric hindrance. As it cuts, the Tn5 transposase also inserts sequencing adapters that allow for rapid library construction by PCR and next-generation sequencing of genome-wide accessible chromatin1,2.
ATAC-seq has become the preferred method to determine regions of chromatin accessibility due to the relatively simple and fast protocol, quality and range of information that can be determined from its results, and small amount of starting material required. Compared to DNase-seq3 (which also measures genome-wide chromatin accessibility), MNase-seq4 (which determines nucleosome positions in open genome regions), and the formaldehyde-mediated FAIRE-seq5, ATAC-seq is faster, cheaper, and more reproducible1. It is also more sensitive, working with starting material of as few as 500 nuclei, compared to the 50 million nuclei required for DNase-seq3. ATAC-seq also has the ability to provide more information about chromatin architecture than other methods, including regions of transcription factor binding, nucleosome positioning, and open chromatin regions1. Effective, single-cell ATAC-seq protocols have been validated, providing information on chromatin architecture at the single-cell level6,7.
ATAC-seq has been used to characterize chromatin architecture across a wide spectrum of research and cells types, including plants8, humans9, and many other organisms. It has also been critical in identifying epigenetic regulation of disease states7. However, the most widely used ATAC-seq protocol includes the major drawback of contamination sequencing reads from mitochondrial DNA. In some data sets, this contamination level can be as high as 60% of sequencing results1. There is a concerted effort in the field to reduce these contaminating mitochondrial reads in order to allow for the more efficient application of ATAC-seq7,10,11.&#160;Here we present an improved ATAC-seq protocol that reduces the mitochondrial DNA contamination rate to just 3%, allowing reduction of around 50% in sequencing costs10. This is made possible by a streamlined process of CD4+ lymphocyte isolation and activation and an improved lysis buffer that is critical in minimizing mitochondrial DNA contamination.
This modifiedATAC-seq protocol has been validated with a wide range of primary cells, including human primary peripheral blood mononuclear cells (PBMCs)10, human primary monocytes, and mouse dendritic cells (unpublished). It has also been used successfully to interrogate melanoma cell lines in a clustered regularly interspaced short palindromic repeats (CRISPR) screen of non-coding elements11. Additionally, the data analysis package described in this protocol and provided on GitHub provides new and experienced researchers with tools to analyze ATAC-seq data. ATAC-seq is the most effective assay to map chromatin accessibility across an entire genome, and modifications to the existing protocol that are introduced here will allow researchers to produce high-quality data with low mitochondrial DNA contamination, reducing sequencing costs and improving ATAC-seq throughput.

<table>
	<tbody>
		<tr>
			<td>1X PBS, Sterile</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>5810/5810&#160;R Swing Bucket Refrigerated Centrifuge with 50 mL, 15 mL, and 1.5 mL Tube Buckets</td>
			<td>Eppendorf</td>
			<td>22625501</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>96 Well Round Bottom Plate</td>
			<td>Thermo Scientific</td>
			<td>163320</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Agilent 4200 Tape Station System</td>
			<td>Agilent</td>
			<td>G2991AA</td>
			<td>Suggested for quality assessment.</td>
		</tr>
		<tr>
			<td>Cryotubes</td>
			<td>Thermo Scientific</td>
			<td>374081</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D8418</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Dynabeads Human T-Activator CD3/CD28</td>
			<td>Invitrogen Life Technologies</td>
			<td>11131D</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Dynabeads Untouched Human CD4 T Cells Kit</td>
			<td>Invitrogen Life Technologies</td>
			<td>11346D</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Dynamagnet</td>
			<td>Invitrogen Life Technologies</td>
			<td>12321D</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Gemini Bio Products</td>
			<td>100-500</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>High Sensitivity DNA Kit</td>
			<td>Agilent</td>
			<td>50674626</td>
			<td>Suggested for quality assessment.</td>
		</tr>
		<tr>
			<td>Magnesium Chloride, Hexahydrate, Molecular Biology Grade</td>
			<td>Sigma</td>
			<td>M2393</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>MinElute PCR Purification Kit (Buffer PB, Buffer PE, Elution Buffer)</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Mr. Frosty</td>
			<td>Thermo Scientific</td>
			<td>5100-0001</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>NaCl, Molecular Biology Grade</td>
			<td>Sigma</td>
			<td>S3014</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>NEBNext High Fidelity 2X PCR Master Mix</td>
			<td>New England BioLabs</td>
			<td>M0541</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Nextera DNA Library Preparation Kit (2X TD Buffer, Tn5 Enzyme)</td>
			<td>Illumina</td>
			<td>FC1211030</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Nuclease Free Sterile dH20</td>
			<td>Gibco</td>
			<td>10977015</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween20)</td>
			<td>Sigma</td>
			<td>P9416</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>PowerUp SYBR Green Master Mix</td>
			<td>Applied Biosystems</td>
			<td>A25780</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Precision Water Bath</td>
			<td>Thermo Scientific</td>
			<td>TSGP02</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Invitrogen Life Technologies</td>
			<td>Q32851</td>
			<td>Suggested for quality assessment.</td>
		</tr>
		<tr>
			<td>Qubit FlouroMeter</td>
			<td>Invitrogen Life Technologies</td>
			<td>Q33226</td>
			<td>Suggested for quality assessment.</td>
		</tr>
		<tr>
			<td>Rosette Sep Human CD4+ Density Medium</td>
			<td>Stem Cell Technologies</td>
			<td>15705</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>Rosette Sep Human CD4+ Enrichment Cocktail</td>
			<td>Stem Cell Technologies</td>
			<td>15022</td>
			<td>Critical Component</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Sterile Resevoir</td>
			<td>Thermo Scientific</td>
			<td>8096-11</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>T100 Thermocycler with Heated Lid</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td>Can use other comparable products.</td>
		</tr>
		<tr>
			<td>Tris-HCL</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>Can use other comparable products.</td>
		</tr>
	</tbody>
</table>

atac-seq assay with low mitochondrial dna contamination from primary human cd4+ t lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible chromatin. In this paper we present an improved ATAC-seq protocol that reduces contaminating mitochondrial DNA reads. While previous ATAC-seq protocols have struggled with an average of 50% contaminating mitochondrial DNA reads, the optimized&#160;lysis buffer introduced in this protocol reduces mitochondrial DNA contamination to an average of 3%. This improved ATAC-seq protocol allows for a near 50% reduction in the sequencing cost. We demonstrate how these high-quality ATAC-seq libraries can be prepared from activated CD4+ lymphocytes, providing step-by-step instructions from CD4+ lymphocyte isolation from whole blood through data analysis. This improved ATAC-seq protocol has been validated in a wide range of cell types and will be of immediate use to researchers studying chromatin accessibility.

From 15 mL of fresh whole blood, this protocol generates an average of 1 million CD4+ T cells. These can be frozen for later processing or used immediately. Viability of the CD4+ T cells, fresh or thawed, was consistently &#62;95%. This method of CD4+ T cell isolation allows for flexibility in source material and collection time. This improved ATAC-seq protocol produces a final library of greater than 1 ng/&#181;L for sequencing. Quality control performed using commercially available syst...

Watch this Scientific Journal Video about ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes at JoVE.com

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

This modified ATAC-seq protocol introduces a new lysis buffer that decreases contaminating mitochondrial reads from 50%to less than 3%The decreased mitochondrial contamination will allow researchers to perform more efficient ATAC-seq with a 50%decrease in sequencing costs. We have validated this new ATAC-seq protocol in human primary PBMCs, human primary monocytes, mouse dendritic cells, and melanoma cell lines. We believe it will be effective in a wide range of cell types.Minimizing sample loss during the cell activation and selection process is critical for obtaining high-quality results with ATAC-seq. We will demonstrate how to handle small numbers of cells from precious samples using basic laboratory equipment. To begin, thaw one vial of previously isolated CD4+T cells in a 37-degree Celsius water bath until the ice has just melted.Then gently transfer the cells to a 15-milliliter conical tube containing nine milliliters of pre-warmed complete RPMI. Centrifuge the cells at 1, 500 times G, and four degrees Celsius, for six minutes. Then discard the supernatant, and gently resuspend the pellet in two milliliters of complete RPMI.After repeating the centrifugation, discard the supernatant, and gently resuspend the cells in 0.5 milliliters of complete RPMI. Use a hemocytometer to count the cells. Using complete RPMI, adjust the cell suspension density to plate 50, 000 CD4+cells in 200 microliters per well of a 96-well round-bottom plate.Then, to prepare magnetic beads, aliquot 12.5 microliters of Human T-Activator CD3/CD28 beads per ATAC-seq sample to a 1.5 milliliter tube. Add one milliliter of 1x PBS to the tube, and place it on a magnet for one minute to wash the beads. Carefully remove and discard the clear supernatant, and remove the tube from the magnet.Resuspend the beads in 13 microliters of complete RPMI per ATAC-seq sample. To activate the CD4+T cells with the adjusted concentration, collect 500, 000 cells in 2.1 milliliters of complete RPMI medium in a 15-milliliter conical tube. Add 12.5 microliters of Human T-Activator beads, prepared and washed in previous steps, and gently mix by inverting the tube.Then gently transfer the cells, with beads, to a sterile reservoir. Using a multi-channel pipette, plate 200 microliters of cells with beads per well of a round-bottom 96-well plate, and incubate in a 37-degree Celsius, 5%CO2 incubator for 48 hours. After complete incubation, centrifuge the plate at 423 times G, and room temperature, for eight minutes.Remove 100 microliters of medium from each well, and collect it in a conical tube before discarding, in order to confirm no bead loss. Then resuspend the cell pellet in the remaining 100 microliters of medium, and collect everything in a 1.5-milliliter tube. To isolate CD4+cells, add 50 microliters of pre-washed CD4 conjugated beads to 500, 000 cells in one milliliter of complete RPMI, and invert to mix.Incubate for 20 minutes at four degrees Celsius, while hand-flicking every six minutes to mix. Place the tube on the magnet for two minutes, until supernatant is clear, and then remove and discard. Remove the tube from the magnet.Wash the bead-bound cells by resuspending them in one milliliter of PBS, plus 2%FCS, and place the tube back on the magnet for one minute. Discard the clear supernatant, and repeat this process for a total of three washes. After the final wash, place the tube on the magnet for one minute.Remove it, discard the supernatant, and place the pellet on ice. Next, to perform nuclei isolation, resuspend the beads and cells in 50 microliters of cold lysis buffer, and centrifuge immediately at 500 G, four degrees Celsius, for 10 minutes. Remove and discard the supernatant.To perform the transposase reaction, resuspend the isolated nuclei pellet with 50 microliters of Tn5 transposase mix. Incubate in a thermocycler for 30 minutes at 37 degrees Celsius, with the lid set to 40 degrees Celsius, and then hold at four degrees Celsius when complete. Immediately after thermocycling, perform a quick bench-top centrifuge spin-down.Place the tube on the magnet for one minute to remove the beads from the product. Without removing the tube from the magnet, transfer the clear supernatant to a DNA purification column. Wash the column once with 250 microliters of PB buffer, and twice with 750 microliters of PE buffer.Then add 10 microliters of elution buffer to elute the sample. Set up the initial PCR amplification reaction in a nuclease-free PCR tube. Place the PCR tube into the thermocycler, and run the PCR amplification program.Then complete the final PCR amplification of the remaining 45 microliters of PCR reaction, as described in the manuscript. Place the PCR tube in the thermocycler and run the program. After complete amplification, purify the libraries using a PCR cleanup kit, following the manufacturer's protocol, eluting in 25 microliters of the elution buffer.Sequence the prepared libraries on a next-generation sequencer to an average read depth of 42 million reads per sample. This improved ATAC-seq protocol produced a final library of greater than one nanogram per microliter for sequencing. Quality control showed DNA fragments between 200 and 1, 000 base pairs, and further sequencing was performed with these high-quality libraries.Libraries prepared following this protocol showed only 3%mitochondrial contamination. The high percentage of usable reads is sufficiently constant across biological replicates. This protocol provided highly reproducible results across technical replicates, as well as biological replicates.Additionally, this protocol took 48 hours, rather than one week or more as previously described, resulting in consistent and efficient activation, as demonstrated by reproducible sequencing results. Furthermore, predicted ATAC-seq peaks were accurately called by the analysis pipeline. Sequencing result analysis revealed clear changes in chromatin state during human T cell activation.Differentially accessible regions of open chromatin were identified between six samples before and 48 hours after activation. Make sure the nuclei lysis reaction is performed with chilled reagents and materials, in order to maximize the recovery of intact nuclei. Our modified nuclei lysis buffer is gentler on the cells, and minimizes contaminating mitochondrial DNA.We look forward to this modified lysis buffer being applied to single-nuclei ATAC-seq as well. We know this protocol will allow researchers to produce higher-quality ATAC-seq data at a decreased cost, helping to forward the epigenetic field. The protocol is straightforward, rapid, and does not require any hazardous reagents or instruments, making it easily accessible for those new to ATAC-seq.

atac-seq-assay-with-low-mitochondrial-dna-contamination-from-primary

Research

JoVE Journal

Genetics

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene ...

Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample material. This was done by targeting anatomical sampling locations previously determined to yield the highest amounts of ancient DNA (aDNA) in a comparative analysis of DNA recovery across the skeleton. Prior research has suggested that these protocols maximize the chances for the successful recovery of ancient human and pathogen DNA from archaeological remains. DNA yields were previously assessed by Parker et al. 2020 in a broad survey of aDNA preservation across multiple skeletal elements from 11 individuals recovered from the medieval (radiocarbon dated to a period of circa (ca.) 1040-1400 CE, calibrated 2-sigma range) graveyard at Krakauer Berg, an abandoned medieval settlement near Pei&#223;en Germany. These eight sampling spots, which span five skeletal elements (pars petrosa, permanent molars, thoracic vertebra, distal phalanx, and talus) successfully yielded high-quality ancient human DNA, where yields were significantly greater than the overall average across all elements and individuals. Yields were adequate for use in most common downstream population genetic analyses. Our results support the preferential use of these anatomical sampling locations for most studies involving the analyses of ancient human DNA from archaeological remains. Implementation of these methods will help to minimize the destruction of precious archaeological specimens.

The protocol presents a series of best practice protocols for the collection of bone powder from eight recommended anatomical sampling locations (specific locations on a given skeletal element) across five different skeletal elements from medieval individuals (radiocarbon dated to a period of ca. 1040-1400 CE, calibrated 2-sigma range).

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample ...

Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that are incorporated during DNA synthesis in human cells. However, this technique has a limited resolution of a few thousand kilobases. Consequently, post-replicative single-stranded DNA (ssDNA) gaps as small as a few hundred bases are not detectable by the standard assay. Here, we describe a modified version of the DNA fiber assay that utilizes the S1 nuclease, an enzyme that specifically cleaves ssDNA. In the presence of post-replicative ssDNA gaps, the S1 nuclease will target and cleave the gaps, generating shorter tracts that can be used as a read-out for ssDNA gaps on ongoing forks. These post-replicative ssDNA gaps are formed when damaged DNA is replicated discontinuously. They can be repaired via mechanisms uncoupled from genome replication, in a process known as gap-filling or post-replicative repair. Because gap-filling mechanisms involve DNA synthesis independent of the S phase, alterations in the DNA fiber labeling scheme can also be employed to monitor gap-filling events. Altogether, these modifications of the DNA fiber assay are powerful strategies to understand how post-replicative gaps are formed and filled in the genome of human cells.

Here we describe two modifications of the DNA fiber assay to investigate single-stranded DNA gaps in replicating DNA after lesion induction. The S1 fiber assay enables the detection of post-replicative gaps using the ssDNA-specific S1 endonuclease, while the gap-filling assay allows visualization and quantification of gap repair.

The DNA fiber assay is a simple and robust method for the analysis of replication fork dynamics, based on the immunodetection of nucleotide analogs that ...

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin accessibility profiling. This technique has been useful for understanding the regulatory mechanisms of gene expression in a range of biological processes. Although ATAC-seq has been modified for different types of samples, there have not been effective modifications of ATAC-seq methods for adipose tissues. Challenges with adipose tissues include the complex cellular heterogeneity, large lipid content, and high mitochondrial contamination. To overcome these problems, we have developed a protocol that allows adipocyte-specific ATAC-seq by employing fluorescence-activated nucleus sorting with adipose tissues from the transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse. This protocol produces high-quality data with minimal wasted sequencing reads while reducing the amount of nucleus input and reagents. This paper provides detailed step-by-step instructions for the ATAC-seq method validated for the use of adipocyte nuclei isolated from mouse adipose tissues. This protocol will aid in the investigation of chromatin dynamics in adipocytes upon diverse biological stimulations, which will allow for novel biological insights.

We present a protocol for assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) specifically on adipocytes using nucleus sorting with adipose tissues isolated from transgenic reporter mice with nuclear fluorescence labeling.