Question: Are neutrophils and other granulocytes compatible with the Fixed RNA Profiling Assay?
Answer: We have performed testing in-house with neutrophils or other granulocytes in the Fixed RNA Profiling Assay (i.e., Single Cell Gene Expression Flex assay) following the Blood Fixation and Cell Isolation for Chromium Fixed RNA Profiling Demonstrated Protocol (CG000721). 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.
Whole blood samples should be collected in vacutainer tubes containing K2 EDTA. DO NOT use any other anticoagulant vacutainer to avoid loss of monocytes. After collection, 1 ml of blood can be added to a tube containing 4 ml Fixation Buffer and fixed at RT overnight (20-24 h) or at 4°C for up to 1 week. After fixation, proceed immediately to Post-Fixation Processing and Leukocyte Isolation (Option B) or store at -80°C after resuspending in 50% glycerol for a final concentration of 10% as described in CG000721.
Once cell isolation is complete, proceed immediately to the appropriate Chromium Fixed RNA Profiling protocols or store the sample after resuspending in appropriate reagents. Samples can be stored at −80ºC for up to 6 months following the guidance provided in the appendix of CG000721.
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.
Figure 1: tSNE plot, Barcode Rank Plot, and Median Gene per Cell for leukocytes isolated via ACK lysis of RBCs (as described above)*
* Forced to 4,500 cells detected
Products: Fixed RNA Profiling Gene Expression, Single Cell Gene Expression Flex