Question: How do I remove granulocytes from my sample?
Answer: Granulocytes can show up in ATAC data and take up sequencing reads. See: How do granulocytes affect my ATAC data?
You may choose to remove granulocytes from PBMC and BMMC samples to clean up the background signal and maximize your sequencing reads. Granulocyte DNA can be removed by:
1) DNase treatment: DNase I will degrade any ambient DNA within the single-cell suspension. This is non-specific to granulocytes. DNase treatment may not be as thorough as sorting out granulocytes but provides an alternative when FACS is not available. Instructions for DNase treatment can be found in the appendix of the Nuclei Isolation for Single Cell ATAC Sequencing demonstrated protocol.
2) Sorting out granulocytes: Granulocytes can be removed via FACs using scatter. Using a 100um nozzle, and no stain, singlets are first gated to remove debris and clumps. Granulocytes have a high side scatter and can be easily gated. Only monocytes and lymphocytes are collected in 1x PBS+0.4% BSA. Sorted cells can then undergo nuclei isolation as described in the Nuclei Isolation for Single Cell ATAC Sequencing demonstrated protocol.
Product: Single Cell ATAC