ATAC-Seq Optimization for Cancer Epigenetics Research

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 cancer^2,3,4. 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 "cut and paste" 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 (<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.

Protokół

1. Preparations before beginning the experiment

1. 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 buffer^12,13 for each cell type using trypan blue staining.
   1. 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.
   2. 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.
2. 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.
3. Turn on the centrifuge to let it reach 4 °C.

2. Cell harvest

1. Aspirate media from a culture of cells at <80% confluency. Cells are typically grown in a 35 mm or 60 mm dish, based on the number of cells needed, treatments, etc.
2. Wash the cells with 1 mL or 3 mL of PBS.
3. 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.
4. 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.
5. Aspirate the media and resuspend the cells 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.
6. Count the cells using a cell counter.
   NOTE: In this study, an automated cell counter (Table of Materials) was used.
7. 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).
8. 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 cells, decrease the cold PBS volume to prepare the cell solution at the same concentration (1 x 10⁶ cells/mL).

3. Cell lysis

1. Transfer 25 µL (= 2.5 x 10⁴ cells) to a new 1.7 mL microcentrifuge tube. Centrifuge for 5 min at 500 x g at 4 °C and carefully discard the supernatant
   NOTE: A swinging bucket rotor is recommended to minimize cell loss. Leave about 3 µL of supernatant to ensure that the cells are not being discarded.
2. Add 25 µL of lysis buffer and resuspend the cells by gentle pipetting up and down. Incubate on ice for 5 min
3. Centrifuge for 5 min at 500 x g at 4 °C and carefully discard the supernatant.
   NOTE: A swinging bucket rotor is recommended to minimize cell loss. Leave about 3 µL of supernatant to ensure that the cells are not being discarded.

4. Tn5 tagmentation

1. Immediately resuspend the pellet in 25 µL of Tn5 reaction mixture. The Tn5 reaction mixture is as follows: 12.5 µL of 2x Tagmentation DNA buffer (TD buffer), 1.25-5 µL of Tn5 transposase, and 11.25-7.5 µL of nuclease-free water.
   NOTE: The standard concentration of Tn5 is 2.5 µL for 2.5 x 10⁴ cells.
2. Incubate for 30 min at 37 °C.
   ​NOTE: Mix the solution every 10 min.

5. DNA purification

NOTE: Purification is required before amplification. DNA purification is done using the MinElute PCR purification kit (Table of Materials).

1. Add 5 µL of 3 M sodium acetate (pH 5.2).
2. Immediately add 125 µL of Buffer PB and mix well.
3. Follow the PCR purification kit protocol starting at step 2.
   1. Place the spin column in a 2 mL collection tube.
   2. Apply the sample to the spin column and centrifuge for 1 min at 17,900 x g at RT.
   3. Discard the flow-through and place the spin column back in the same collection tube.
   4. Add 750 µL of Buffer PE to the spin column and centrifuge for 1 min at 17,900 x g at RT.
   5. Discard the flow-through and place the spin column back in the same collection tube.
   6. Centrifuge the spin column for 5 min to dry the membrane completely.
   7. Discard the flow-through and place the spin column in a new 1.7 mL microcentrifuge tube.
   8. Elute the DNA fragments with 10 µL of Buffer EB.
   9. Let the column stand for 1 min at RT.
   10. Centrifuge for 1 min at 17,900 x g at RT to elute the DNA.

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.

1. Set up the following PCR reaction in 200 µL PCR tubes (50 µL for each sample). The PCR reaction mixture is as follows: 10 µL of eluted DNA (step 5.), 2.5 µL of 25 mM Adapter 1, 2.5 µL of 25 mM Adapter 2, 25 µL of 2x PCR master mix, and 10 µL of nuclease-free water
2. 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.
3. Real-time qPCR (total of 14.5 µL per sample)
   1. Using 5 µL 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.
   2. Prepare the PCR reaction mixture as follows: 5 µL of PCR product from step 6.2., 0.75 µL of SYBR gold (1000x diluted), 5 µL of 2x PCR master mix, and 3.75 µL of nuclease-free water.
      ​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 µL of SYBR Gold (stock concentration 10,000x) was added to 999 µL of nuclease-free water. The run time of RT-qPCR mentioned in Table 3 is about 60 min.
4. Determine the required number of additional PCR cycles using the run parameters from step 6.3.
   1. Number of cycles = 1/4 maximum (saturated) fluorescence intensity (typically 3 or 4 PCR cycles)
5. PCR amplification program part 2 (volume = 45 µL; 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.)

7. Beads purification

NOTE: Here, AMPure XP (Table of Materials) beads were used.

1. To the PCR products amplified in the previous step, add 150 µL of beads at RT.
   NOTE: Homemade AMPure beads¹⁴ can be used in this process.
2. Incubate for 15 min at RT.
3. Place the tubes on a magnetic stand for 5 min.
4. Carefully remove the supernatant.
5. Wash the beads with 200 µL of 80% ethanol.
   NOTE: 80% ethanol should be prepared fresh.
6. Repeat step 7.4.
7. Remove the ethanol completely.
8. Air dry the samples for 10 min at RT.
9. Resuspend with 50 µL of elution buffer (10 mM Tris-HCL pH 8.0).
10. Repeat the beads purification (steps 7.1.-7.9.) to further minimize primer contamination.

8. DNA concentration and quality check

1. Measure the DNA concentration by using a nucleic acid quantification kit (Table of Materials).
2. 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).

9. Sequencing

1. Perform high-throughput sequencing using the amplified DNA samples^12,13.
   ​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.

10. Data analysis

1. Data filtration based on base call quality: Set the minimum average base quality score to 20 (99% accuracy).
2. 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.
3. Genome mapping: Map the reads to the appropriate genome with Bowtie using the following parameters "-I 0 -X 2000 -m 1"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%
4. Deduplication: Mark the PCR duplicates by Picard tools¹⁶.
5. 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.

Wyniki

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...

Dyskusje

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...

Materiały

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