Question: How do granulocytes affect my ATAC (standalone or Multiome) data?
Answer: Granulocytes, specifically neutrophils, can be present in PBMC and BMMC samples. Activated Neutrophils can participate in NETosis, a process in which they form neutrophil extracellular traps (NETs) , complexes of proteins and chromatin that trap pathogens. During NETosis, neutrophils experience chromatin swelling, in which their chromatin decondenses and eventually bursts out of the cell . NETosis results in lysed neutrophil cells with lots of open chromatin.
Transposition of samples with lots of activated neutrophils with lots of open chromatin can result in:
- A high abundance of neutrophil-specific fragments in the final library-the chromatin in NETs is very open and efficiently fragmented during transposition
- A high fraction of short, mononucleosome protected fragments-the chromatin in NETs is very open and efficiently fragmented during transposition
- Libraries with overall low targeting metrics-these fragments are coming from all regions of the genome, and will not overlap with called peaks from other cells.
As a result, the resulting ATAC final library distribution will show an increase in the mononucleosome fragments and the ATAC targeting plot will show a distinct population at the bottom right-hand corner. Neutrophil transposed fragments will colocalize with dead/dying cells and are filtered out by Cell Ranger ATAC 1.2.0 software.
If desired, granulocytes can be removed via FACs or a DNase treatment can be performed to reduce the ambient DNA present in NETs. See: How do I remove granulocytes from my sample?
If granulocytes are not removed during sample preparation, they will be transposed and sequenced. These reads will be filtered out as non-cells during analysis by Cell Ranger ATAC 1.2.
Recently, other publications have demonstrated the added benefit of sorting out granulocytes for single-cell ATAC assays: https://pubmed.ncbi.nlm.nih.gov/33835024/
Products: Single Cell ATAC, Single Cell Multiome ATAC + GEX