How should samples with high neutrophil (or granulocyte) content be handled, and why is neutrophil removal recommended?

Answer:
Granulocyte depletion is strongly recommended before nuclei isolation. The proportion of granulocytes in the final input sample should ideally be below 5%.

The main concern is activated neutrophils undergoing NETosis (neutrophil extracellular trap formation). During this process, chromatin becomes highly decondensed, nuclear membranes rupture, and large amounts of accessible chromatin fragments are released into the suspension.

These free chromatin fragments can substantially interfere with genuine ATAC signals, resulting in:

• Elevated background noise 
• Increased nonspecific accessibility signals 
• Reduced ATAC library quality 
• Lower downstream data interpretability 

High-risk sample types commonly enriched for granulocytes include:

• PBMC samples from certain inflammatory or disease conditions containing low-density granulocytes 
• Purified neutrophil (PMN) samples 
• Lung tissues and bronchoalveolar lavage fluid 
• Subcutaneous tumors, gastric tissues, and intestinal tissues, especially inflammatory or tumor samples 
• Bone and bone marrow 
• Blood-rich body fluids such as cerebrospinal fluid or drainage fluid 

Recommended depletion strategies:

Operational recommendations:

• Granulocyte depletion should always be performed before nuclei isolation to minimize contamination from ruptured nuclei.
• CD45 negative selection alone is usually insufficient for efficient neutrophil removal and is therefore not recommended as the primary depletion strategy.