Dec 25, 2025
ATAC-seq library preparation in cultured cells
- Xin Xu1,
- Yujuan Wang1
- 1Princess Margaret Cancer Centre
Protocol Citation: Xin Xu, Yujuan Wang 2025. ATAC-seq library preparation in cultured cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj1rrnvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 21, 2025
Last Modified: December 25, 2025
Protocol Integer ID: 235615
Keywords: ATAC-seq, chromatin accessibility, epigenomics, open chromatin, transposase, sequencing library preparation, cancer cells, seq library preparation, library preparation in cultured cell, accessible chromatin, dna purification, atac, cells this protocol, including nuclei preparation, seq, cultured cell, nuclei preparation, transposition reaction
Disclaimer
This protocol is provided for research use only and may require optimization depending on experimental conditions.
Abstract
This protocol describes assay for transposase-accessible chromatin using sequencing (ATAC-seq) in cultured cells, including nuclei preparation, transposition reaction, DNA purification, and library amplification.
Materials
- ATAC-Resuspension buffer (ATAC-RSB)**: 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, supplemented with 0.1% NP-40, 0.1% Tween-20, 0.01% digitonin
- Transposition master mix**: 2x TD buffer (20 mM Tris-HCl, pH 7.6; 10 mM MgCl2; 20% dimethylformamide)
- Tn5 transposase (Illumina, 20034197),
- 1% digitonin (Millipore, 300410),
- 10% Tween-20 (Sigma/Roche cat# 11332465001)
- MinElute PCR purification kit (QIAGEN, 28004)
- 1x NEBNext PCR Master Mix**
Troubleshooting
Safety warnings
- Cell number and lysis conditions may require optimization depending on cell type.
- Over-lysis can reduce library complexity.
- Accurate pipetting during transposition is critical for reproducibility.
Before start
- Perform all steps on ice or at 4°C unless otherwise indicated.
- Use freshly prepared buffers or thawed aliquots kept on ice.
- Count viable cells accurately prior to starting the assay.
- Pre-chill centrifuges and rotators.
- Prepare transposition master mix immediately before use.
Cell preparation and lysis
3m
Collect 50,000 viable cells per sample.
Resuspend cells in 50 µL cold ATAC-Resuspension buffer (ATAC-RSB) containing:
10 mM Tris-HCl (pH 7.4)
10 mM NaCl
3 mM MgCl2
Supplemented with:
0.1% NP-40
0.1% Tween-20
0.01% digitonin
Incubate cell suspension on ice for 3 min.
3m
Wash the lysate with 1 mL of cold ATAC-RSB without NP-40 and digitonin.
Pellet nuclei by centrifugation at 500 xg and carefully remove supernatant.
Transposition reaction
30m
Resuspend nuclei in 50 µL transposition master mix containing:
2x TD buffer (20 mM Tris-HCl, pH 7.6; 10 mM MgCl2; 20% dimethylformamide)
2.5 µL Tn5 transposase (Illumina, 20034197)
16.5 µL PBS
0.5 µL 1% digitonin (Millipore, 300410)
0.5 µL 10% Tween-20
Incubate transposition reaction at 37°C for 30 min with 1,000 rpm mixing.
30m
DNA purification
30m
Purify transposed DNA fragments using the MinElute PCR purification kit (QIAGEN, 28004) according to the manufacturer’s instructions.
30m
Elute purified DNA in 20 µL nuclease-free water or elution buffer.
Library amplification
2h
Construct sequencing libraries using 1x NEBNext PCR Master Mix and custom primers as described by Buenrostro et al.
1h
Amplify libraries according to standard ATAC-seq PCR conditions.
1h
Protocol references
Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).
Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).