Experimental Design Considerations

Replication Strategy

Plan for at least 3-4 biological replicates per condition. ATAC-seq can show high variability between samples, making adequate replication crucial for detecting reproducible accessible chromatin regions and statistical power in downstream analysis.

Quality Control Before Sequencing

• Cell viability: Ensure >90% viability before nuclei isolation
• Nuclei integrity: Check for intact nuclei without cytoplasmic contamination
• Library complexity: Assess fragment size distribution on Bioanalyzer - expect nucleosome laddering pattern
• Enrichment validation: Verify enrichment at known accessible regions (e.g., TSS) using qPCR

Spike-in Normalization

Consider spike-in controls when comparing samples with potentially different numbers of accessible sites (e.g., developmental stages, drug treatments). Drosophila chromatin or synthetic DNA standards can help normalize for technical variation and enable quantitative comparisons across conditions.

Sequencing Depth

Target 25-50 million paired-end reads per sample for mammalian genomes. Higher depth may be needed for detecting condition-specific peaks or working with limited cell numbers.

ENCODE Guidelines

Follow the ENCODE ATAC-seq pipeline for standardized data processing and quality metrics. Key benchmarks include TSS enrichment >7, fraction of reads in peaks >0.3, and library complexity measures.

Data Processing Workflow

[Link to your analysis pipeline documentation]