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 MgCl2, and 0.1% NP-40.</li>
		<li>To prepare CSK buffer, mix 10 mM PIPES pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, and 0.1% Triton X-100.</li>
	</ol>
	</li>
	<li>Ensure that the cell culture conditions are optimal; examine the cells under a light microscope to make sure that there are no elevated levels of apoptosis.</li>
	<li>Turn on the centrifuge to let it reach 4 &#176;C.</li>
</ol>
2. Cell harvest
<ol>
	<li>Aspirate media from a culture of cells at &#60;80% confluency. Cells are typically grown in a 35 mm or 60 mm dish, based on the number of cells needed, treatments, etc.</li>
	<li>Wash the cells with 1 mL or 3 mL of PBS.</li>
	<li>Add 0.5 mL or 1 mL of Trypsin and incubate for 2-3 min (depending on the cell types). Carefully use a 1 mL pipette to mix well.</li>
	<li>Add 1 mL or 2 mL of media to the plate and mix well. Transfer to a 15 mL conical tube. Centrifuge the cells in a 15 mL tube for 3 min at 300 x g. 
	NOTE: A swinging bucket rotor is recommended to minimize cell loss.</li>
	<li>Aspirate the media and resuspend the cells&#160;in 1 mL or 2 mL of medium. 
	NOTE: It is critical to prepare a homogenous single-cell solution. For tissues, a Dounce homogenizer can be used to homogenize tissues and minimize large cell clumps or aggregates. Some tissues require filtration (e.g., cell strainers) and/or FACS cell sorting to isolate viable cells and obtain a single cell solution.</li>
	<li>Count the cells using a cell counter. 
	NOTE: In this study, an automated cell counter (Table of Materials) was used.</li>
	<li>Centrifuge the required cell volume (e.g., a volume containing 1 x 106 cells based on cell count) at 300 x g for 3 min at room temperature (RT).</li>
	<li>Resuspend the cell pellet containing 1 x 106 cells in 1 mL of cold PBS. 
	NOTE: If cell numbers are less than 1 x 106&#160;cells, decrease the cold PBS volume to prepare the cell solution at the same concentration (1 x 106&#160;cells/mL).</li>
</ol>
3. Cell lysis
<ol>
	<li>Transfer 25 &#181;L (= 2.5 x 104 cells) to a new 1.7 mL microcentrifuge tube. Centrifuge&#160;for 5 min at 500 x g at 4 &#176;C and carefully discard the supernatant 
	NOTE: A swinging bucket rotor is recommended to minimize cell loss. Leave about 3 &#181;L of supernatant to ensure that the cells are not being discarded.</li>
	<li>Add 25 &#181;L of lysis buffer and resuspend the cells by gentle pipetting up and down. Incubate on ice for 5 min</li>
	<li>Centrifuge for 5 min at 500 x g at 4 &#176;C and carefully&#160;discard the supernatant. 
	NOTE: A swinging bucket rotor is recommended to minimize cell loss. Leave about 3 &#181;L of supernatant to ensure that the cells are not being discarded.</li>
</ol>
4. Tn5 tagmentation
<ol>
	<li>Immediately resuspend the pellet in 25 &#181;L of Tn5 reaction mixture. The Tn5 reaction mixture is as follows: 12.5 &#181;L of 2x Tagmentation DNA buffer (TD buffer), 1.25-5 &#181;L of Tn5 transposase, and 11.25-7.5 &#181;L of nuclease-free water. 
	NOTE: The standard concentration of Tn5 is 2.5 &#181;L for 2.5 x 104 cells.</li>
	<li>Incubate for 30 min at 37 &#176;C. 
	&#8203;NOTE: Mix the solution every 10 min.</li>
</ol>
5. DNA purification
NOTE: Purification is required before amplification. DNA purification is done using the MinElute PCR purification kit (Table of Materials).
<ol>
	<li>Add 5 &#181;L of 3 M sodium acetate (pH 5.2).</li>
	<li>Immediately add 125 &#181;L of Buffer PB and mix well.</li>
	<li>Follow the PCR purification kit protocol starting at step 2.
	<ol>
		<li>Place the spin column in a 2 mL collection tube.</li>
		<li>Apply the sample to the spin column and centrifuge for 1 min at 17,900 x g at RT.</li>
		<li>Discard the flow-through and place the spin column back in the same collection tube.</li>
		<li>Add 750 &#181;L of Buffer PE to the spin column and centrifuge for 1 min at 17,900 x g at RT.</li>
		<li>Discard the flow-through and place the spin column back in the same collection tube.</li>
		<li>Centrifuge the spin column for 5 min to dry the membrane completely.</li>
		<li>Discard the flow-through and place the spin column in a new 1.7 mL microcentrifuge tube.</li>
		<li>Elute the DNA fragments with 10 &#181;L of Buffer EB.</li>
		<li>Let the column stand for 1 min at RT.</li>
		<li>Centrifuge for 1 min at 17,900 x g at RT to elute the DNA.</li>
	</ol>
	</li>
</ol>
6. PCR amplification
NOTE: PCR amplification of transposed (tagmented) DNAs is necessary for sequencing. Nextera kit adapters (Table of Materials) were used in this example. The primers used in this study are listed in Table 1.
<ol>
	<li>Set up the following PCR reaction in 200 &#181;L PCR tubes (50 &#181;L for each sample). The PCR reaction mixture is as follows: 10 &#181;L of eluted DNA (step 5.), 2.5 &#181;L of 25 mM Adapter 1, 2.5 &#181;L of 25 mM Adapter 2, 25 &#181;L of 2x PCR master mix, and 10 &#181;L of nuclease-free water</li>
	<li>Perform PCR amplification program part 1 (Table 2). 
	NOTE: For initial amplification, the same conditions are used for all samples using a standard thermal cycler.</li>
	<li>Real-time qPCR (total of 14.5 &#181;L per sample)
	<ol>
		<li>Using 5 &#181;L of the product from the PCR amplification part 1 (step 6.2.), set up the following reaction, this time including SYBR gold and detection in a real-time PCR machine.</li>
		<li>Prepare the PCR reaction mixture as follows: 5 &#181;L of PCR product from step 6.2., 0.75 &#181;L of SYBR gold (1000x diluted), 5 &#181;L of 2x PCR master mix, and 3.75 &#181;L of nuclease-free water. 
		&#8203;NOTE: The precise cycle numbers for library amplification before reaching saturation for each sample are determined by qPCR (Table 3) to reduce the GC and size bias in PCR10. SYBR Gold was diluted with nuclease-free water. To make the diluted solution, 1 &#181;L of SYBR Gold (stock concentration 10,000x) was added to&#160;999 &#181;L of nuclease-free water. The run time of RT-qPCR mentioned in Table 3 is about 60 min.</li>
	</ol>
	</li>
	<li>Determine the required number of additional PCR cycles using the run parameters from step 6.3.
	<ol>
		<li>Number of cycles = 1/4 maximum (saturated) fluorescence intensity (typically 3 or 4 PCR cycles)</li>
	</ol>
	</li>
	<li>PCR amplification program part 2 (volume = 45 &#181;L; Table 4): Once you determine the cycle number, set up PCRs with additional cycles calculated at step 6.4.1. For instance, if the cycle number calculated is 3, set up 3 cycles in the program below. The total cycles would be 8 (5 in step 6.2., plus 3 additional cycles in step 6.5.)</li>
</ol>
7. Beads purification
NOTE: Here, AMPure XP (Table of Materials) beads were used.
<ol>
	<li>To the PCR products amplified in the previous step, add 150 &#181;L of beads at RT. 
	NOTE: Homemade AMPure beads14 can be used in this process.</li>
	<li>Incubate for 15 min at RT.</li>
	<li>Place the tubes on a magnetic stand for 5 min.</li>
	<li>Carefully remove the supernatant.</li>
	<li>Wash the beads with 200 &#181;L of 80% ethanol. 
	NOTE: 80% ethanol should be prepared fresh.</li>
	<li>Repeat step 7.4.</li>
	<li>Remove the ethanol completely.</li>
	<li>Air dry the samples for 10 min at RT.</li>
	<li>Resuspend with 50 &#181;L of elution buffer (10 mM Tris-HCL pH 8.0).</li>
	<li>Repeat the beads purification (steps 7.1.-7.9.) to further minimize primer contamination.</li>
</ol>
8. DNA concentration and quality check
<ol>
	<li>Measure the DNA concentration by using a nucleic acid quantification kit (Table of Materials).</li>
	<li>Check the amplified DNA fragments by gel electrophoresis using SYBR Gold (0.5x TBE, 1.5% agarose gel) or an automated electrophoresis system (e.g., TapeStation, bioanalyzer).</li>
</ol>
9. Sequencing
<ol>
	<li>Perform high-throughput sequencing using the amplified DNA samples12,13. 
	&#8203;NOTE: Amplified DNA samples are ready for high-throughput sequencing. To retain nucleosomal DNA fragment information, paired-end sequencing is recommended over single-end sequencing. Both ends of the DNA fragments indicate where the transposase bind. For mapping open chromatin regions, 30 million reads/sample is typically sufficient.</li>
</ol>
10. Data analysis
<ol>
	<li>Data filtration based on base call quality: Set the minimum average base quality score to 20 (99% accuracy).</li>
	<li>Adapter trimming: Remove the adapter sequences using an appropriate tool. 
	NOTE: The Trim Galore wrapper tool (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) was used to remove adapter sequences. For ATAC-seq libraries prepared by Illumina Tn5, the following sequence is used as an adapter sequence: CTGTCTCTTATACACATCT. The minimum sequence length to recognize adapter contamination is set to 5.</li>
	<li>Genome mapping: Map the reads to the appropriate genome with Bowtie using the following parameters &#34;-I 0 -X 2000 -m 1&#34;15. An example of mapping quality from MDA-MB-231 basal breast cancer cells is below: 
	Reads with at least one reported alignment: 52.79% 
	Reads that failed to align: 13.10% 
	Reads with alignments suppressed due to -m: 34.11%</li>
	<li>Deduplication: Mark the PCR duplicates by Picard tools16.</li>
	<li>Generating a genome coverage file for data visualization: Convert the deduplicated paired-end reads to single-end read fragments. Genome coverage tracks are obtained using genomeCoverageBed from bedtools17.</li>
</ol>

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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><tbody><tr><td>1.5 mL microcentrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td>Natural</td></tr><tr><td>10 &amp;micro;L XL TipOne tips</td><td>USA Scientific</td><td>1120-3810</td><td>Filtered and low-retention</td></tr><tr><td>100 &amp;micro;L XL TipOne RPT tips</td><td>USA Scientific</td><td>1182-1830</td><td>Filtered and low-retention</td></tr><tr><td>100 &amp;micro;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 &amp;micro;L TipOne RPT tips</td><td>USA Scientific</td><td>1183-1810</td><td>Filtered and low-retention</td></tr><tr><td>200 &amp;micro;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, &amp;ge;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&amp;amp;D Systems</td><td>S10350</td><td>Triple 0.1 &amp;micro;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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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, &amp;ge;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-optimization-for-cancer-epigenetics-research

Research

JoVE Journal

Cancer Research

4.2K Views.  University of North Dakota School of Medicine and Health Sciences. 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. 

Video: ATAC-Seq Optimization for Cancer Epigenetics Research

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 ...

Genetics

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

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.

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 ...

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin accessibility profiling. This technique has been useful for understanding the regulatory mechanisms of gene expression in a range of biological processes. Although ATAC-seq has been modified for different types of samples, there have not been effective modifications of ATAC-seq methods for adipose tissues. Challenges with adipose tissues include the complex cellular heterogeneity, large lipid content, and high mitochondrial contamination. To overcome these problems, we have developed a protocol that allows adipocyte-specific ATAC-seq by employing fluorescence-activated nucleus sorting with adipose tissues from the transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mouse. This protocol produces high-quality data with minimal wasted sequencing reads while reducing the amount of nucleus input and reagents. This paper provides detailed step-by-step instructions for the ATAC-seq method validated for the use of adipocyte nuclei isolated from mouse adipose tissues. This protocol will aid in the investigation of chromatin dynamics in adipocytes upon diverse biological stimulations, which will allow for novel biological insights.

We present a protocol for assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) specifically on adipocytes using nucleus sorting with adipose tissues isolated from transgenic reporter mice with nuclear fluorescence labeling.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a robust technique that enables genome-wide chromatin ...

CATCH-UP: A High-Throughput Upstream-Pipeline for Bulk ATAC-Seq and ChIP-Seq Data

Assay for transposase-accessible chromatin (ATAC) and chromatin immunoprecipitation (ChIP), coupled with next-generation sequencing (NGS), have revolutionized the study of gene regulation. A lack of standardization in the analysis of the highly dimensional datasets generated by these techniques has made reproducibility difficult to achieve, leading to discrepancies in the published, processed data. Part of this problem is due to the diverse range of bioinformatic tools available for the analysis of these types of data. Secondly, a number of different bioinformatic tools are required sequentially to convert raw data into a fully processed and interpretable output, and these tools require varying levels of computational skills. Furthermore, there are many options for quality control that are not uniformly employed during data processing. We address these issues with a complete assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) upstream pipeline (CATCH-UP), an easy-to-use, Python-based pipeline for the analysis of bulk ChIP-seq and ATAC-seq datasets from raw fastq files to visualizable bigwig tracks and peaks calls. This pipeline is simple to install and run, requiring minimal computational knowledge. The pipeline is modular, scalable, and parallelizable on various computing infrastructures, allowing for easy reporting of methodology to enable reproducible analysis of novel or published datasets.

ATAC-seq and ChIP-seq allow detailed investigation of gene regulation; however, processing these data types is challenging and often inconsistent between research groups. We present CATCH-UP: an easy-to-use computational pipeline that allows standardized and reproducible data processing and analysis of new and published ATAC/ChIP-seq datasets.

Assay for transposase-accessible chromatin (ATAC) and chromatin immunoprecipitation (ChIP), coupled with next-generation sequencing (NGS), have ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

Bioengineering

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Biology

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

Neuroscience

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 ...

Immunology and Infection

3' End Sequencing Library Preparation with A-seq2

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3&#39;UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3&#39; end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3&#39; end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3&#39; ends in a wide range of conditions, from 106 or fewer cells.

This protocol describes a method for mapping pre-mRNA 3&#39; end processing sites.

ATAC-Seq Optimization for Cancer Epigenetics Research

Summary

Explore More Videos

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

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

Adipocyte-Specific ATAC-Seq with Adipose Tissues Using Fluorescence-Activated Nucleus Sorting

CATCH-UP: A High-Throughput Upstream-Pipeline for Bulk ATAC-Seq and ChIP-Seq Data

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

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

3' End Sequencing Library Preparation with A-seq2