Feb 18, 2026
Bulk ATAC-seq
- Lexi Bounds1,
- Alejandro Barrera1,
- Maria ter Weele1,
- Siyan Liu1,
- Euphy Wu2,
- Shengyu Li1,
- Revathy Venukuttan1,
- Ruhi Rai1,
- Wancen Mu2,
- Nahid Iglesias1,
- Paola Giusti-Rodriguez3,
- Timothy Reddy1,
- Yun Li2,
- Raluca Gordan2,
- Andrew Allen2,
- Michael Love2,
- Patrick Sullivan2,
- Greg Crawford1,
- Charles Gersbach1
- 1Duke University;
- 2University of North Carolina;
- 3University of Florida
- Gersbach Lab
Protocol Citation: Lexi Bounds, Alejandro Barrera, Maria ter Weele, Siyan Liu, Euphy Wu, Shengyu Li, Revathy Venukuttan, Ruhi Rai, Wancen Mu, Nahid Iglesias, Paola Giusti-Rodriguez, Timothy Reddy, Yun Li, Raluca Gordan, Andrew Allen, Michael Love, Patrick Sullivan, Greg Crawford, Charles Gersbach 2026. Bulk ATAC-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp12kdgzp/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: February 17, 2026
Last Modified: February 18, 2026
Protocol Integer ID: 243537
Keywords: bulk atac, using neural progenitor cell, neural progenitor cell, npc, seq, seq method, seq this protocol
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from the work of Lexi Bounds in the Gersbach lab at Duke University.
Abstract
This protocol describes bulk ATAC-seq methods and analysis using Neural Progenitor Cells (NPCs).
Materials
KAPA Library Quantification Kit (Roche #7960255001)
High Sensitivity DNA ScreenTape Analysis (Agilent #5067)
Troubleshooting
Bulk ATAC-seq
100,000 NPCs are harvested using Accutase and resuspended in 1 mL of cold ATAC-seq resuspension buffer and the Omni-ATAC seq protocol was used to generate ATAC-sequencing libraries (Ref. 1).
Following pre-amplification, 3 additional cycles of PCR were performed prior to final amplification and cleanup.
Library concentrations and sizes were obtained using the KAPA Library Quantification Kit (Roche #7960255001) and High Sensitivity DNA ScreenTape Analysis (Agilent #5067).
Perform ATAC-seq for two biological replicates. The final libraries are pooled to 3 nM and sequenced on a HiSeq 4000 with 1x50bp configuration.
Bulk ATAC-seq analysis
The pipeline for processing and analysis of the bulk ATAC-seq data can be found here: https://github.com/Duke-GCB/GGR-cwl/blob/master/v1.0/ATAC-seq_pipeline/pipeline-se-blacklist-removal.cwl.
Quality control of FASTQ files is performed using FASTQC (Ref.2).
Adapter reads are then trimmed using Trimmomatic.
Reads are then aligned to the reference genome using Bowtie (Ref. 3).
Duplicate reads are removed using Picard MarkDuplicates and ENCODE hg38 blacklist reads were removed using BEDtools2 (Ref. 4).
Peak calling is performed using MACS2 (Ref. 5) with narrowPeak settings.
Sequencing-depth normalized ATAC bigWig files were generated using deeptools (Ref. 6) bamCoverage.
Protocol references
1. Corces, M. R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017).
2. Babraham Bioinformatics - FastQC A Quality Control tool for High Throughput Sequence Data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
3. Langmead, B. Aligning short sequencing reads with Bowtie. Curr. Protoc. Bioinforma. Chapter 11, Unit 11.7 (2010).
4. Quinlan, A. R. BEDTools: The Swiss-Army Tool for Genome Feature Analysis. Curr. Protoc. Bioinforma. 47, 11.12.1-34 (2014).
5. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
6. Ramírez, F., Dündar, F., Diehl, S., Grüning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187-191 (2014).