We gratefully acknowledge the UND Genomics Core facility for outstanding technical assistance.
This work was funded by the National Institutes of Health [P20GM104360 to M.T., P20 GM104360 to A.D.] and start-up funds provided by the University of North Dakota School of Medicine and Health Sciences, Department of Biomedical Sciences [to M.T.].

1. Preparations before beginning the experiment
<ol>
	<li>Prepare lysis buffer stock. 
	NOTE: The optimal nuclear isolation buffer can be different for each cell type. We recommend testing both the hypotonic buffer used in the original paper8 and a CSK buffer12,13 for each cell type using trypan blue staining.
	<ol>
		<li>To prepare hypotonic buffer (Greenleaf buffer), mix 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgC...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+integrator+complex+attenuates+promoter-proximal+transcription+at+protein-coding+genes.">The integrator complex attenuates promoter-proximal transcription at protein-coding genes.</a> Molecular Cell. 76 (5), 738-752 (2019).</li><li>Corces, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+ATAC-seq+protocol+reduces+background+and+enables+interrogation+of+frozen+tissues.">An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.</a> Nature Methods. 14 (10), 959-962 (2017).</li><li>Porter, J. 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The authors declare that there are no relevant or material financial interests that relate to the research described in this paper.

ATAC-seq has been widely used for mapping open and active chromatin regions. Cancer cell progression is frequently driven by genetic alterations and epigenetic reprogramming, resulting in altered chromatin accessibility and gene expression. An example of this reprogramming is seen during the epithelial-to-mesenchymal transition (EMT) and its reverse process, mesenchymal-to-epithelial transition (MET), which are known to be key cellular reprogramming processes during tumor metastasis30. Another exa...

Chromatin accessibility is a key requirement for the regulation of gene expression on a genome-wide scale1. Changes in chromatin accessibility are frequently associated with several disease states, including cancer2,3,4. Over the years, numerous techniques have been developed to enable researchers to probe the chromatin landscape by mapping regions of chromatin accessibility. Some of them include DNase-seq (DNase I hypersensitive sites sequencing)5, FAIRE-seq (formaldehyde-assisted isolation of regulatory elements)6, MAPit (methyltransferase accessibility protocol for individual templates)7, and the focus of this paper, ATAC-seq (assay for transposase-accessible chromatin)8. DNase-seq maps accessible regions by employing a key feature of DNase, namely the preferential digestion of naked DNA free from histones and other proteins such as transcription factors5. FAIRE-seq, similar to ChIP-seq, utilizes formaldehyde crosslinking and sonication, except no immunoprecipitation is involved, and the nucleosome-free regions are isolated by phenol-chloroform extraction6. The MAPit method uses a GC methyltransferase to probe chromatin structure at single-molecule resolution7. ATAC-seq relies on the hyperactive transposase, Tn58. The Tn5 transposase preferentially binds to open chromatin regions and inserts sequencing adapters into accessible regions. Tn5 operates through a DNA-mediated &#34;cut and paste&#34; mechanism, whereby the transposase preloaded with adapters binds to open chromatin sites, cuts DNA, and ligates the adapters8. Tn5 bound regions are recovered by PCR amplification using primers that anneal to these adapters. FAIRE-seq and DNase-seq require a large amount of starting material (~100,000 cells to 225,000 cells) and a separate library preparation step before sequencing9. On the other hand, the ATAC-seq protocol is relatively simple and requires a small number of cells (&#60;50,000 cells)10. Unlike the FAIRE-seq and DNase-seq techniques, the sequencing library preparation of ATAC-seq is relatively easy, as the isolated DNA sample is already being tagged with the sequencing adapters by Tn5. Therefore, only the PCR amplification step with appropriate primers is needed to complete the library preparation, and the prior processing steps such as end-repair and adapter ligation need not be performed, thus saving time11. Secondly, ATAC-seq avoids the need for bisulfite conversion, cloning, and amplification with region-specific primers required for MAPit7. Due to these advantages, ATAC-seq has become a hugely popular method for defining open chromatin regions. Although the ATAC-seq method is simple, multiple steps require optimization to obtain high-quality and reproducible data. This manuscript discusses optimization procedures for standard ATAC-seq library preparation, especially highlighting three parameters: (1) lysis buffer composition, (2) Tn5 transposase concentration, and (3) cell number. In addition, this paper provides example data from the optimization conditions using both cancerous and non-cancerous adherent epithelial cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>Natural</td>
		</tr>
		<tr>
			<td>10 &#181;L XL TipOne tips</td>
			<td>USA Scientific</td>
			<td>1120-3810</td>
			<td>Filtered and low-retention</td>
		</tr>
		<tr>
			<td>100 &#181;L XL TipOne RPT tips</td>
			<td>USA Scientific</td>
			<td>1182-1830</td>
			<td>Filtered and low-retention</td>
		</tr>
		<tr>
			<td>100 &#181;L XL TipOne tips</td>
			<td>USA Scientific</td>
			<td>1120-1840</td>
			<td>Filtered and low-retention. Beveled Grade</td>
		</tr>
		<tr>
			<td>15 mL Conical Centrifuge Tubes</td>
			<td>Corning</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L TipOne RPT tips</td>
			<td>USA Scientific</td>
			<td>1183-1810</td>
			<td>Filtered and low-retention</td>
		</tr>
		<tr>
			<td>200 &#181;L TipOne RPT tips</td>
			<td>USA Scientific</td>
			<td>1180-8810</td>
			<td>Filtered and low-retention</td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tubes</td>
			<td>Fisherbrand</td>
			<td>06-443-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>YBP136010</td>
			<td>Genetic Analysis Grade</td>
		</tr>
		<tr>
			<td>All the cell lines used in this study are obtained from ATCC</td>
			<td>ATCC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Allegra X-30R Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>364658</td>
			<td>SX2415</td>
		</tr>
		<tr>
			<td>AMPure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Bead purification kit</td>
		</tr>
		<tr>
			<td>CellDrop Cell Counter</td>
			<td>DeNovix</td>
			<td>CellDrop FL</td>
			<td>Cell counter</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>MilliporeSigma</td>
			<td>EDS</td>
			<td>BioUltra, anhydrous, &#8805;99% (titration)</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>MilliporeSigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>ThermoFisher Scientific</td>
			<td>AC615100020</td>
			<td>Anhydrous; Fisher Scientific - Decon Labs Sterilization Products</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - TET Tested</td>
			<td>R&#38;D Systems</td>
			<td>S10350</td>
			<td>Triple 0.1 &#181;m filtered</td>
		</tr>
		<tr>
			<td>Gibco DMEM 1x</td>
			<td>ThermoFisher Scientific</td>
			<td>11965092</td>
			<td>[+] 4.5 g/L D-glucose; [+] L-Glutamine; [-] Sodium pyruvate</td>
		</tr>
		<tr>
			<td>Gibco PBS 1x</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td>pH 7.4</td>
		</tr>
		<tr>
			<td>Gibco Trypsin-EDTA 1x</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td>(0.25%), phenol red</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>IBI Scientific</td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>MilliporeSigma</td>
			<td>G8898</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>MilliporeSigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>MilliporeSigma</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen Qubit Fluorometer</td>
			<td>ThermoFisher Scientific</td>
			<td>Q32857</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>MilliporeSigma</td>
			<td>M3634</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>DNA purification kit</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>IBI Scientific</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>MilliporeSigma</td>
			<td>S8045</td>
			<td>BioXtra, &#8805;98% (acidimetric), pellets (anhydrous)</td>
		</tr>
		<tr>
			<td>NEBNext High-Fidelity 2x PCR Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0541</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera DNA Sample Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-121-1030</td>
			<td>2x TD and Tn5 Transposase</td>
		</tr>
		<tr>
			<td>NP - 40 (IGEPAL CA-630)</td>
			<td>MilliporeSigma</td>
			<td>I8896</td>
			<td>for molecular biology</td>
		</tr>
		<tr>
			<td>PCR Detection System</td>
			<td>BioRad</td>
			<td>1855484</td>
			<td>CFX384 Real-Time System. C1000 Touch Thermal Cycler</td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>MilliporeSigma</td>
			<td>P1851</td>
			<td>BioPerformance Certified, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay kit</td>
			<td>ThermoFisher Scientific</td>
			<td>Q32854</td>
			<td>Invitrogen; Nucleic acid quantitation kit</td>
		</tr>
		<tr>
			<td>Quibit Assay Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>Q32856</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>MilliporeSigma</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Homemade</td>
			<td>-</td>
			<td>pH 5.2</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>IBI Scientific</td>
			<td>57-50-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Gold</td>
			<td>ThermoFisher Scientific</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green Supermix, 1.25 mL</td>
			<td>BioRad</td>
			<td>1708882</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>TempAssure 0.2 mL PCR 8-Tube Strips</td>
			<td>USA Scientific</td>
			<td>1402-4700</td>
			<td>Flex-free, natural, polypropylene</td>
		</tr>
		<tr>
			<td>TempPlate 384-WELL PCR PLATE</td>
			<td>USA Scientific</td>
			<td>1438-4700</td>
			<td>Single notch. Natural polypropylene</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>MilliporeSigma</td>
			<td>648311</td>
			<td>ULTROL Grade</td>
		</tr>
		<tr>
			<td>Triton x-100</td>
			<td>IBI Scientific</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TrueSeq Dual Index Sequencing Primer Kit</td>
			<td>Illumina</td>
			<td>PE-121-1003</td>
			<td>paired-end</td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>Q32851</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>MilliporeSigma</td>
			<td>P7949</td>
			<td>BioXtra, viscous liquid</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>MilliporeSigma</td>
			<td>W3500</td>
			<td>sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
	</tbody>
</table>

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.

To obtain successful and high-quality ATAC-seq data, it is important to optimize the experimental conditions. ATAC-seq library preparation can be separated into the five major steps (Figure 1), namely cell lysis, tagmentation (fragmentation and adapter insertion by Tn5), genomic DNA purification, PCR amplification, and data analysis. As an initial process, the cell lysis (nuclear isolation) buffer must be first optimized for each cell type. Either the hypotonic buffer described in the origin...

Watch this Scientific Journal Video about ATAC-Seq Optimization for Cancer Epigenetics Research at JoVE.com

ATAC-Seq Optimization for Cancer Epigenetics Research

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 ...

ATAC-seq allows us to identify the status of the open chromatin regions in the genome, which is often altered in the disease states compared to the normal condition. Fewer cell input, a simplified protocol of Tn5 digestion, followed by isolation of fragmented DNA and library preparation by PCR amplification and shorter experimental time make it a more advantageous technique. Comparison of the altered epigenetic landscape by ATAC-seq between normal and disease conditions can shed light on the disease-associated chromatin regulatory elements and be useful for designing new therapeutic strategies.This technique is easy to perform, as it does not include any critical experimental steps. To get successful ATAC-seq results, it only requires a couple of standardization steps. To begin, transfer 25 microliters from a cell suspension containing one million cells per milliliter in PBS to 1.7-milliliter microcentrifuge tube and centrifuge at 500 G for five minutes at four degrees Celsius.Carefully discard the supernatant, and add 25 microliters of lysis buffer. Re-suspend the cells by gently pipetting up and down, and incubate on ice for five minutes. Centrifuge at 500 G for five minutes at four degrees Celsius, and carefully discard the supernatant.Immediately re-suspend the pellet in 25 microliters of Tn5 reaction mixture, and incubate for 30 minutes at 37 degrees Celsius. Mix the solution every 10 minutes. Add five microliters of 3-molar sodium acetate and 125 microliters of buffer PB to the Tn5 tagmented DNA solution.Mix well. Next, place the spin column in a 2-milliliter collection tube, and apply the sample to the spin column. Centrifuge at 17, 900 G for one minute at room temperature, and discard the flow-through.Add PE buffer to the column, and spin the column. Place the spin column back in the same collection tube, and centrifuge it for five minutes to dry the membrane completely. Discard the flow-through, and place the spin column in a new 1.7-milliliter microcentrifuge tube.Elute the DNA fragments with 10 microliters of buffer EB, and let the column stand for one minute at room temperature. Then centrifuge at 17, 900 G for one minute at room temperature to elute the DNA. Set up the PCR reaction in 200-microliter PCR tubes, and perform PCR Amplification Program Part One.For real-time QPCR, prepare the PCR reaction mixture by adding five microliters of PCR product, 0.75 microliters of 1, 000-times-diluted SYBR Gold, five microliters of 2X PCR Master Mix and 3.75 microliters of nuclease-free water. Determine the required number of additional PCR cycles using the run parameters. Once the cycle number is determined, set up PCRs with the additional cycles calculated earlier.To the amplified PCR products, add 150 microliters of beads, and incubate for 15 minutes at room temperature. Place the tubes on a magnetic stand for five minutes, and carefully remove the supernatant. Wash the beads with 200 microliters of 80%ethanol, and remove the supernatant.Remove the ethanol completely, and air dry the samples for 10 minutes at room temperature. Finally, re-suspend with 50 microliters of elution buffer. Place the tube on a magnetic stand, then transfer the eluate to a fresh 1.7-milliliter microcentrifuge tube.A comparison of cell lysis efficiency using trypan blue is shown here. NMuMG cells were treated with a hypotonic buffer and CSK buffer and stained with trypan blue. Higher cell lysis efficiency was observed in CSK-treated cells.ATAC-seq libraries were prepared from MDA-MB-231 breast cancer cells. After PCR amplification, PCR products were analyzed on a 0.5X TBE, 1.5%agarose gel before and after beads purification. Primers are mostly removed by the initial beads purification.DNA band patterns from 1X TAE 1%agarose gel electrophoresis and an automated electrophoresis are also indicated. SYBR Gold was used to visualize the DNA fragments shown here. A genome browser track showing the ERS1 locus is presented here.The genome coverage of the ATAC-seq data in T47D cells is shown in this image. Primers from a previous publication were used, and their target regions are highlighted in yellow. A bar graph depicting primer amplicon enrichment in ATAC-seq libraries is shown here.ATAC-seq libraries were prepared by the hypotonic buffer or a CSK buffer. The representative image shows the genome browser visualization for ATAC-seq optimization. The ATAC-seq data from the 25, 000-cell input condition showed the highest enrichment of nucleosome-free fragments, while the 100, 000-cell input condition presented the highest mononucleosomal DNAs.Genome browser tracks showing ATAC-seq data from different cell numbers are presented here. HOMER peak annotation analysis is depicted in this representative image. HOMER classified each peak as a promoter proximal or distal peak.The number of cell input choice of cell lysis buffer and the concentration of the Tn5 enzyme dependent on the cell type are the most critical parameter that must be standardized. Tn5 fused with protein A in the cut-and-tack technique is widely used to identify DNA binding site for a protein of interest using antibodies specific to that protein of interest.

atac-seq-optimization-for-cancer-epigenetics-research

Research

JoVE Journal

Cancer Research

3.7K Views.  University of North Dakota School of Medicine and Health Sciences. ATAC-seq allows us to identify the status of the open chromatin regions in the genome, which is often altered in the disease states compared to the normal condition. Fewer cell input, a simplified protocol of Tn5 digestion, followed by isolation of fragmented DNA and library preparation by PCR amplification and shorter experimental time make it a more advantageous technique. Comparison of the altered epigenetic landscape by ATAC-seq between normal and disease conditions can shed light on the ...