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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	<li>Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+native+chromatin+for+fast+and+sensitive+epigenomic+profiling+of+open+chromatin,+DNA-binding+proteins+and+nucleosome+position.">Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.</a> Nature Methods. 10 (12), 1213-1218 (2013).</li><li>Buenrostro, J. D., Wu, B., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATAC-seq:+A+Method+for+Assaying+Chromatin+Accessibility+Genome-Wide.">ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide.</a> Current Protocols in Molecular Biology. 21, 1-29 (2015).</li><li>Song, L., Crawford, G. 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 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&#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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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

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 ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

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, ...

Lentiviral Mediated Gene Silencing in Human Pseudoislet Prepared in Low Attachment Plates

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, the data obtained from rodent islets are often not fully reproduced in or applicable to human islets due to well-known differences in islet structure and function between the species. Currently, techniques that are available to manipulate gene expression of human islets are very limited. Introduction of transgene into intact islets by adenovirus, plasmid, and oligonucleotides often suffers from low efficiency and high toxicity. Low efficiency is especially problematic in gene downregulation studies in intact islets, which require high efficiency. It has been known that enzymatically-dispersed islet cells reaggregate in culture forming spheroids termed pseudoislets. Size-controlled reaggregation of human islet cells creates pseudoislets that maintain dynamic first phase insulin secretion after prolonged culture and provide a window to efficiently introduce lentiviral short hairpin RNA (shRNA) with low toxicity. Here, a detailed protocol for the creation of human pseudoislets after lentiviral transduction using two commercially available multiwell plates is described. The protocol can be easily performed and allows for efficient downregulation of genes and assessment of dynamism of insulin secretion using human islet cells. Thus, human pseudoislets with lentiviral mediated gene modulation provide a powerful and versatile model to assess gene function within human islet cells.

A protocol to create gene modified human pseudoislets from dispersed human islet cells that are transduced by lentivirus carrying short hairpin RNA (shRNA) is presented. This protocol utilizes readily available enzyme and culture vessels, can be performed easily, and produces genetically modified human pseudoislets suitable for functional and morphological studies.

Various genetic tools are available to modulate genes in pancreatic islets of rodents to dissect function of islet genes for diabetes research. However, ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are two types of HM based on microscopic morphological evaluation, complete HM (CHM) and partial HM (PHM). These can be further subdivided based on the parental contribution to the molar genomes. Such characterization of HM, by morphology and genotype analyses, is crucial for patient management and for the fundamental understanding of this intriguing pathology. It is well documented that morphological analysis of HM is subject to wide interobserver variability and is not sufficient on its own to accurately classify HM into CHM and PHM and distinguish them from hydropic non-molar abortions. Genotyping analysis is mostly performed on DNA and tissues from formalin-fixed paraffin-embedded (FFPE) products of conception, which have less than optimal quality and may consequently lead to wrong conclusions. In this article, detailed protocols for multiplex genotyping and flow cytometry analyses of FFPE molar tissues are provided, along with the interpretation of the results of these methods, their troubleshooting, and integration with the morphological evaluation, p57KIP2 immunohistochemistry, and fluorescence in situ hybridization (FISH) to reach a correct and robust diagnosis. Here, the authors share the methods and lessons learned in the past 10 years from the analysis of approximately 400 products of conception.

Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results&#8217; interpretation and integration.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are ...