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 requirements. Scale the protocol as needed. Pre-warm phosphate buffered saline (PBS) + 2% fetal calf serum (FCS) to room temperature (RT) and adjust the centrifuge to RT before starting the CD4+ T cell enrichment procedure.
<ol>
	<li>Add 750 &#181;L of human CD4+ T cell enrichment cocktail to 15 mL of whole blood in a 50 mL conical tube and mix gently by inversion. Incubate at RT for 20 min. When incubation is complete, add 15 mL of PBS + 2% FCS to the tube and mix gently by inversion.</li>
	<li>Prepare a fresh 50 mL conical tube with 15 mL of density medium. Carefully layer the diluted blood sample onto the top of the density medium, being sure not to disrupt the density medium/blood interface that forms. Centrifuge for 20 min at 1,200 x g and RT with acceleration set to 1 and the descending brake OFF. 
	NOTE: It is critical to set the acceleration to 1 and descending brake OFF on the centrifuge to avoid disruption of the cell layer in the density medium.</li>
	<li>Collect the enriched CD4+ T cells from the density medium/plasma interface using a narrow stem transfer pipette. Transfer the collected cells to a fresh 50 mL conical tube. Centrifuge the collected CD4+ T cells for 8 min at 423 x g and RT. 
	NOTE: Make sure to return the centrifuge acceleration to 9 and descending brake to ON for step 1.3 and all of the following steps.</li>
	<li>Discard the supernatant and wash the cell pellet twice with 50 mL of PBS + 2% FCS, centrifuging for 8 min at 423 x g and RT. Discard the final supernatant wash and suspend the washed cell pellet in 2 mL of PBS + 2% FCS.</li>
	<li>Count cells using a hemocytometer. To continue with ATAC-seq, proceed to step 2.3. To freeze cells for later processing, proceed to step 1.6.</li>
	<li>Add 1 mL of fresh freezing medium (90% FCS + 10% dimethyl sulfoxide) per 1 million cells. Aliquot 1 mL of cells in freezing medium in cryogenic safe tubes. Place tubes at -80 &#176;C overnight in a slow-freezing container. The next day, transfer tubes to liquid nitrogen for long-term storage. 
	NOTE: 1 million CD4+ T cells will be isolated per 2 mL of original volume of whole blood. Freeze 500,000 cells in 1 mL aliquots of freezing medium. Make fresh freezing medium at every use.</li>
</ol>
2. Activate and Purify CD4+ T Cells
NOTE: This rapid protocol for activating and purifying CD4+ T cells only requires 48 h and results in 95% viable, activated CD4+ T cells. Cool the centrifuge to 4 &#176;C before beginning the protocol.
<ol>
	<li>Thaw 1 vial (500,000) of CD4+ T cells in a 37&#8201;&#176;C water bath until the ice has just melted. Gently transfer cells to a 15 mL conical tube containing 9 mL of pre-warmed Roswell Park Memorial Institute-1640 (RPMI-1640) media supplemented with 10% FCS, hereafter referred to as complete RPMI. 
	NOTE: Do not let cells thaw completely to avoid exposure to DMSO.</li>
	<li>Centrifuge for 6 min at 1500 x g and 4 &#176;C. Discard the supernatant and gently suspend the pellet in 2 mL of complete RPMI. Repeat the spin down, discard the supernatant, and gently suspend cells in 0.5 mL of complete RPMI.</li>
	<li>Count the cells using a hemocytometer. Adjust the density of the cell suspension with complete RPMI to plate 50,000 cells in 200 &#181;L per well of a 96-well round bottom plate. 
	NOTE: From one frozen tube of 500K CD4+ T cells, plate 10 wells of 50,000 CD4+ T cells in 200 &#181;L per well.</li>
	<li>Prepare magnetic beads conjugated with human T-activator anti-CD3 and anti-CD28 antibodies.
	<ol>
		<li>Aliquot 12.5 &#181;L of human T-activator CD3/CD28 beads per ATAC-seq sample to a 1.5 mL tube. Wash the beads with 1 mL of 1x PBS and place the tube on a magnet for 1 min.</li>
		<li>Carefully remove and discard the clear supernatant, remove the tube from magnet, and suspend the beads in 13 &#181;L complete RPMI per ATAC-seq sample. 
		NOTE: Calculate the amount of beads necessary based on the number of ATAC-seq samples being processed. This protocol requires 12.5 &#181;L of CD3/CD28 beads per 500,000 cells, which constitutes one ATAC-seq sample.</li>
	</ol>
	</li>
	<li>Activate the CD4+ T cells. Collect 500,000 cells in 2.1 mL of complete RPMI medium in a 15 mL conical tube and add 12.5 &#181;L of pre-washed human T-activator beads. Mix gently by inverting the tube.</li>
	<li>Gently transfer the cells with beads to a sterile reservoir and plate 200 &#181;L of cells with beads per well of round bottom 96-well plate using a multi-channel pipette. Incubate for 48 h in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Post incubation, centrifuge the 96-well plate for 8 min at 423 x g and RT. Remove 100 &#181;L of medium per well from the 96-well plate, collecting it to a conical tube before discarding to ensure no bead loss. Resuspend the cell pellet in the remaining 100 &#181;L and collect all in a 1.5 mL tube. 
	NOTE: 10 wells of activated T cells will be collected in a 1 mL volume.</li>
	<li>Perform CD4+ isolation. Add 50 &#181;L of pre-washed CD4 conjugated beads to 500,000 cells in 1 mL of complete RPMI. Combine beads and cells by pipette. Incubate for 20 min at 4 &#176;C in the fridge. Agitate beads and cell mixture every 6 min. 
	NOTE: This protocol is to be used for one sample of 500,000 cells. Adjust as necessary for two or more samples of 500,000.</li>
	<li>When the incubation is complete, place the tube on the magnet for 2 min. Remove and discard the supernatant when clear. Wash the bead-bound cells by removing the tube from the magnet, suspending the bead-bound cells in 1 mL of PBS + 2% FCS, replacing the tube on the magnet for 1 min, and discarding the clear supernatant. Repeat this process for a total of 3 washes. 
	NOTE: Be careful not to discard any beads during washes.</li>
	<li>After the final wash, place the tube on the magnet for 1 min. Remove and discard the supernatant and place the pellet on ice. Proceed to section 3 (ATAC-seq).</li>
</ol>
3. ATAC-seq
NOTE: In this step, nuclei are isolated from activated CD4+ T cells for ATAC-seq. The lysis buffer used in this protocol has been improved to be gentler on the nuclei, resulting in more efficient digestion and higher quality results. All centrifugation steps in section 3 are performed with a fixed-angle centrifuge maintained at 4 &#176;C. Cool the centrifuge before beginning protocol.
<ol>
	<li>Perform nuclei isolation. Resuspend beads and cells with cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.03% polysorbate 20). Centrifuge immediately at 500 x g for 10 min at 4 &#176;C. Remove and discard the supernatant.</li>
	<li>Perform the transposase reaction. Suspend the isolated nuclei pellet with 50 &#181;L of Tn5 transposase mix (Table 1). Incubate in a thermocycler for 30 min at 37 &#176;C with a 40 &#176;C lid, holding at 4 &#176;C when complete.</li>
	<li>After thermocycling, immediately perform a quick benchtop centrifuge spin down and place the tube on the magnet for 1 min to remove the beads from the product.</li>
	<li>Transfer the clear supernatant from the tube on the magnet to a DNA purification column. Wash the column once with 250 &#181;L of Buffer PB and twice with 750 &#181;L of Buffer PE. Elute the sample in 10 &#181;L of elution buffer. Proceed directly to step 3.5. 
	NOTE: This step amplifies the DNA fragments with the ligated adaptors, here referred to as the ATAC-seq library. In order to multiplex several ATAC-seq libraries to be run on one NGS lane, use unindexed Primer 1 for all samples and a different indexed Primer 2 for individual samples (Table 2). Primers are used at working concentrations of 1.25 &#181;M.</li>
	<li>Set up the initial PCR amplification reaction in a nuclease-free PCR tube, combining the components in the order and volume specified in Table 3. Place the PCR tube into the thermocycler and run the PCR amplification program with cycling conditions as specified in&#160;Table 4.</li>
	<li>Monitor the reaction by qPCR. Set up the qPCR reaction in a nuclease-free PCR tube, combining the components in the order and amount specified in Table 5. Place in qPCR machine and cycle as specified in Table 6.
	<ol>
		<li>To determine the optimal number of additional amplification cycles for the remaining 45 &#181;L reaction, create a plot with cycle number on the x-axis and relative fluorescence (RFU) on the y-axis. The optimal number of additional amplification cycles is one-third the number of cycles it takes for the qPCR reaction to reach plateau. 
		NOTE: Monitoring the reaction by qPCR allows for determination of the optimal number of PCR cycles desired to obtain optimal library fragment amplification while minimizing GC content and size bias.</li>
	</ol>
	</li>
	<li>Complete the final PCR amplification of the remaining 45 &#181;L of PCR reaction. Place the PCR tube with the amplification reaction from step 3.5 back in the thermocycler and run the program as described in Table 7. 
	NOTE: For samples processed as described in this protocol, the optimal total number of PCR amplification cycles was determined to be 12.</li>
	<li>After the amplification is complete, purify the libraries using a PCR cleanup kit following the manufacturer&#8217;s protocol, eluting in 25 &#181;L of the elution buffer. 
	NOTE: Amplified libraries can be stored at 4 &#176;C for up to 48 h or frozen at -20 &#176;C for long-term storage.</li>
</ol>
4. ATAC-seq Library Quality Analysis
NOTE: It is important to validate the quality and quantity of ATAC-seq libraries before next-generation sequencing. The quality and quantity of the libraries should be assessed using commercially available kits (see Table of Materials).
<ol>
	<li>Assess the quality and quantity of the ATAC-seq libraries using a microfluidics-based platform for sizing, quantification and quality control of DNA. See Figure 2 for representative quality assessment results. 
	NOTE: The concentration of the ATAC-seq libraries must be &#62;1 ng/&#181;L to obtain quality sequencing results. On average, 30 nM in 25 &#181;L was obtained.</li>
</ol>
5. Sequencing and Data Analysis
NOTE: This analysis pipeline allows users to control the quality of reads mapping procedure, adjust coordinates for experimental design, and call peaks for downstream analysis. The following are the commands lines and explanation of execution. The data analysis package is available at (https://github.com/s18692001/bulk_ATAC_seq).
<ol>
	<li>Sequence the prepared libraries on a next generation sequencer to an average read depth of 42 million reads per sample.</li>
	<li>Estimate the quality of sequencing reads by checking files generated by FastQC12 software package using the command: 
	fastqc -o &#60;output_directory&#62; &#60;fastq_file&#62;</li>
	<li>Trim the bases of reads by Trimmomatic13 software, if needed, as determined by the FastQC quality check, using the command (for pair-ended): 
	java -jar &#60;path to trimmomatic jar file&#62; PE &#60;input1&#62; &#60;input2&#62; &#60;paired output1&#62; &#60;unpaired output1&#62; &#60;paired output2&#62; &#60;unpaired output2&#62; HEADCROP:&#60;crop bases&#62;</li>
	<li>Align reads to human reference genome (hg38) by Bowtie214 software: 
	bowtie2 -x &#60;reference genome&#62; -1 &#60;input pair 1&#62; &#60;input pair 2&#62; -S &#60;output SAM file&#62; 
	NOTE: The argument -x is for the basename of the index for the reference genome, and -S is for the SAM format output.</li>
	<li>Check thequality of mapped reads using Samtools15 flagstat package: 
	samtools flagstat &#60;BAM file&#62;</li>
	<li>Sort the mapped reads file and remove duplicates using Picard16 tool: 
	java -jar picard.jar SortSam INPUT=&#60;input SAM file name&#62; OUTPUT=&#60;output sorted BAM file name&#62; SORT_ORDER=coordinate&#8221; and &#8220;java -jar picard.jar MarkDuplicates INPUT=&#60;sorted BAM file&#62; OUTPUT=&#60;output BAM file without duplicates&#62; REMOVE_DUPLICATES=TRUE</li>
	<li>Index BAM file, trim the unused chromosome reads and shift the coordinates for the experimental reason of ATAC-seq17: 
	java -jar picard.jar BuildBamIndex INPUT=&#60;BAM file&#62; 
	samtools idxstats &#60;Input BAM file&#62; | cut -f 1 | grep -v chrY | grep -v chrM | grep -v chrUn | xargs samtools view -b &#60;Input BAM file&#62; &#62; &#60;Output trimmed BAM file&#62; 
	bedtools bamtobed -i &#60;Input trimmed BAM file&#62; &#62; &#60;Output trimmed BED file&#62; 
	awk &#39;BEGIN {OFS = &#34;\t&#34;} ; {if ($6 == &#34;+&#34;) print $1, $2 + 4, $3 + 4, $4, $5, $6; else print $1, $2 - 5, $3 - 5, $4, $5, $6}&#39; &#60;Input trimmed BED file&#62; &#60;Trimmed shifted BED file&#62;</li>
	<li>Delete the reads that are shifted to negative coordination: 
	awk &#39;{if ($2 &#62; 0) print $1 &#34;\t&#34; $2 &#34;\t&#34; $3 &#34;\t&#34; $4 &#34;\t&#34; $5 &#34;\t&#34; $6 }&#39; &#60;Input BED file&#62; &#60;Output non-negative coordination BED file&#62;.</li>
	<li>Convert the BED file to BAM file for following DiffBind18 analysis: 
	bedtools bedtobam -i &#60;Input BED file&#62; -g &#60;reference genome&#62; &#60;Output BAM file&#62;</li>
	<li>Perform peak calling by DiffBind18 software package: 
	macs2 callpeak -t &#60;Input BAM file&#62; -f BAM -g hs -nomodel --nolambda --keep-dup all --call-summits --outdir &#60;Output directory path&#62; -n &#60;Output name&#62; -B -q 0.01 --bdg --shift -100 --extsize 200 
	NOTE: After filtering, expect a median of 37 million reads per sample. Mitochondrial DNA contamination will range from 0.30%&#8211;5.39% (1.96% on average). There will be a low rate of multiply mapped reads (6.7%&#8211;56%, 19% on average) and a relatively high percentage of usable nuclear reads (60%&#8211;92%, 79% on average).</li>
</ol>

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The authors have nothing to disclose.

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&amp;nbsp;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 dH&lt;sub&gt;2&lt;/sub&gt;0</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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

12.5K Views.  Boston University. 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.

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

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 ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

Immunology and Infection

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

Cancer Research

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Author Spotlight: High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Biochemistry

Nuclei Isolation from Mouse Cardiac Progenitor Cells at Single-Cell Resolution

Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene expression and chromatin state of individual cells allows the identification of the cell type and state. Single-cell sequencing approaches have revealed a number of important characteristics of cardiac progenitor cell heterogeneity. However, these methods are generally restricted to fresh tissue, which limits studies with diverse experimental conditions, as the fresh tissue must be processed at once in the same run to reduce the technical variability. Therefore, easy and flexible procedures to produce data from methods such as single-nucleus RNA sequencing (snRNA-seq) and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing (snATAC-seq) are needed in this area. Here, we present a protocol to rapidly isolate nuclei for subsequent single-nuclei dual-omics (combined snRNA-seq and snATAC-seq). This method allows the isolation of nuclei from frozen samples of cardiac progenitor cells and can be combined with platforms that use microfluidic chambers.

Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution  

Here, we present a protocol describing cell nuclei preparation. After microdissection and enzymatic dissociation of cardiac tissue into single cells, the progenitor cells were frozen, followed by isolation of pure viable cells, which were used for single-nucleus RNA sequencing and the single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing analyses.

The developing heart is a complex structure containing various progenitor cells controlled by complex regulatory mechanisms. The examination of the gene ...

Developmental Biology

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Metabolic adaptation is fundamental for T cells as it dictates differentiation, persistence, and cytotoxicity. Here, an optimized protocol for monitoring mitochondrial respiration in ex vivo cytokine-differentiated human primary T cells is presented.

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

Scalable ATAC-seq Protocol for Chromatin Accessibility in Rodent Neutrophils

ATAC-Seq Library Preparation of Murine Bone Marrow-Derived Neutrophils

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and understanding epigenomic regulation. Neutrophils, as a crucial leukocyte type in immune responses, undergo substantial chromatin architectural changes during differentiation and activation, which significantly impact the gene expression necessary for their functions. ATAC-seq has been instrumental in uncovering key transcription factors in neutrophil maturation, revealing pathogen-specific epigenomic signatures, and identifying therapeutic targets for autoimmune diseases. However, neutrophils' sensitivity to the external milieu complicates high-quality ATAC-seq data production. Here, we propose a scalable protocol for preparing ATAC-seq libraries from rodent bone marrow-derived neutrophils, featuring improved immunomagnetic separation to ensure optimal cell viability and high-quality libraries. The vital elements impacting the library quality and optimization principles for methodological extension are discussed in detail. This protocol will support the researchers who are willing to study the chromatin architecture and epigenomic reprogramming of neutrophils, advancing studies in basic and clinical immunology.

This manuscript outlines a protocol for preparing an ATAC-seq library of neutrophils from murine bone marrow, aiming to guarantee optimal neutrophil viability and high library quality. It offers step-by-step instructions on BMC preparation, immunomagnetic sorting, and library construction, serving as a valuable guide, especially for newcomers to studying neutrophils.

Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq) is a powerful, high-throughput technique for assessing chromatin accessibility and ...

Measuring Mitochondrial Function of Na&#239;ve and Effector CD8 T Cells

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. Lymphocyte biology has been explored using bioenergetic analysis and has now become a critically import tool in the field. Thus, we sought to optimize a bioenergetic analysis assay that can be adapted with pretreatments and acute injection for receptor stimulations. Here, we evaluated CD8 T cell ex vivo metabolism using the Cell Mito Stress Test to assess rates of oxygen consumption and extracellular acidification in na&#239;ve and effector CD8 T cells. Antigen-specific effector CD8 T cells were derived via ex vivo stimulation, and na&#239;ve CD8 T cells harvested from splenocytes and isolated with magnetic bead column separation. 
Pretreatments are performed in microplates and we detail how to prepare sensor cartridges. We show how injection ports can loaded with drugs to indirectly measure metabolic capacities and with metabolic modulators, this protocol can be used to study specific enzyme activity. T-cell receptor stimulations can be studied in real time with acute injection and stimulation with anti-CD3/CD28 using the injection ports. Instrument analyzers are used for measurements and data collection and data visualization is done with software programs to interpret cellular metabolism. This strategy produces an extensive amount of data on immune cell biology and mitochondrial bioenergetics allowing researchers to customize the protocol in numerous ways to explore CD8 T cell metabolism.

CD8 T cell bioenergetics can be interrogated using the Mito Stress Test. This methodology can be used to study acute and chronic metabolic programming. This protocol describes approaches to examine the relationships between T cell receptor biology and bioenergetic analysis.

Understanding how immunometabolism impacts the function, differentiation, and fate of lymphocytes has garnered significant interest and attention. ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

Medicine

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...