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to successfully characterize chromatin architecture of human primary PBMCs10, human monocytes, mouse dendritic cells (unpublished), and cultured melanoma cell lines11.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 ...

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