Extensive evaluation of ATAC-seq protocols for native or formaldehyde-fixed nuclei.

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• The Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is a widely used method for measuring chromatin accessibility and studying gene regulation.
• Existing ATAC-seq protocols have numerous variations, but a comprehensive head-to-head evaluation of these choices in a consistent setting is lacking.

Purpose of the Study:

• To systematically evaluate the impact of different ATAC-seq protocol components on data quality and utility.
• To compare various reaction buffers, temperatures, enzyme sources, and nuclei preparation methods (native vs. fixed).

Main Methods:

• Conducted a comprehensive comparison of 24 ATAC-seq experimental conditions by systematically varying major protocol components.
• Utilized a well-characterized cell line and primary mouse lung tissue, with four replicates for each condition.
• Tested multiple input amounts for primary samples.

Main Results:

• Native nuclei generally yielded more peaks than fixed nuclei, with reaction temperature significantly affecting data quality metrics for both.
• Protocol choices, such as buffer type and temperature, influenced data quality metrics inconsistently between native and fixed samples.
• In-house and commercial enzymes performed similarly, while specific buffers (e.g., THS) showed distinct profiles in native samples.

Conclusions:

• The relationship between common library quality metrics varies with temperature and fixation, necessitating the evaluation of multiple metrics.
• ATAC-seq protocol choices can bias the profiling of functional elements, highlighting the need to test several formulations for new experiments.
• The study provides insights for optimizing ATAC-seq workflows, including for crosslinked samples to profile archival clinical specimens.