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...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNase-seq:+a+high-resolution+technique+for+mapping+active+gene+regulatory+elements+across+the+genome+from+mammalian+cells.">DNase-seq: a high-resolution technique for mapping active gene regulatory elements across the genome from mammalian cells.</a> Cold Spring Harbor Protocols. (2), (2010).</li><li>Pajoro, A., Mui&#241;o, J. M., Angenent, G. C., Kaufmann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+Nucleosome+Occupancy+by+MNase-seq:+Experimental+Protocol+and+Computational+Analysis.">Profiling Nucleosome Occupancy by MNase-seq: Experimental Protocol and Computational Analysis.</a> Methods in Molecular Biology. 1675, 167-181 (2018).</li><li>Giresi, P. G., Kim, J., McDaniell, R. M., Iyer, V. R., Lieb, J. 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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to successfully characterize chromatin architecture of human primary PBMCs10, human monocytes, mouse dendritic cells (unpublished), and cultured melanoma cell lines11.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.

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