Question: Are neutrophils and other granulocytes compatible with the Fixed RNA Profiling Assay?
Answer: We have performed very limited testing in-house with neutrophils or other granulocytes in the Fixed RNA Profiling Assay. Fixation of these samples should limit the degradation after collection that is often observed due to high levels of RNases and other inhibitory compounds. Similar considerations exist for sample preparation before fixation, as with other assays.
Neutrophils or other granulocytes can be isolated from whole blood samples using the leukocyte isolation instructions outlined in Option 1 of the Isolation of Leukocytes, Bone Marrow, and Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing Demonstrated Protocol (also shown below). When isolating neutrophils from whole blood, Red Blood Cells should be removed from the sample using an RBC lysis buffer such as ACK (ammonium chloride potassium). Following RBC lysis, neutrophils should be fixed immediately after collection at 4°C overnight (16-24 hr) to limit degradation after collection following the Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling Demonstrated Protocol. For additional sample preparation guidance, please see: Can I process neutrophils (or other granulocytes) using 10x Single Cell applications?
Analysis:
Cell Ranger software performs an unsupervised analysis of the sample. If you have a sample that contains two very distinct sub-populations, one with lots of RNA/cell and another with very little RNA/cell, the latter may be incorrectly labeled as background. Thus, CellRanger may filter out the neutrophils that do not have many mRNAs. We recommend using the --force-cells parameter and setting its value to the target cell recovery.
We also have a tutorial on preserving and annotating the neutrophils in your data.
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex