Question: Can I process neutrophils (or other granulocytes) using 10x Single Cell applications?
Answer: Single-cell analysis of neutrophils, and granulocytes generally, remains a significant challenge. This is because neutrophils (and other granulocytes) have relatively low RNA content and relatively high levels of RNases and other inhibitory compounds. This may result in fewer transcripts detected in GEMs and less usable sequencing reads. Furthermore, neutrophils are particularly sensitive to degradation after collection and cannot be isolated via density gradient centrifugation (as is the case for other nucleated blood cells, often referred to as PBMCs).
A few points to consider when optimizing the 10x Genomics Single-Cell Assays for neutrophils:
1) Immediate processing is critical. Ideally, neutrophils should be processed immediately after collection, and any delay longer than two hours is likely to result in a failed run. Isolation of neutrophils from frozen samples may also present significant challenges and fresh samples are always preferred.
2) Additional wash steps can help remove some of the RNases that are present in the single-cell suspension. This is an option if you have plenty of single cells available. Supplementing RNase inhibitor into the loading buffer may also help preserve your RNA. We typically use RNase inhibitor with nuclei, so we don't expect any adverse effects. We have tested RNase inhibitor (Sigma Aldrich PN-3335399001)
3) Neutrophils may not react well to long incubation periods on ice. They prefer room temperature during the sample prep. This may be an option if you specifically work with enriched neutrophils.
4) Increasing the PCR cycles by 2 extra cycles during cDNA amplification in a non-enriched sample may help to bring the neutrophil-specific transcripts above background.
5) 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). This will reduce the amount of sequencing required by enriching the sample for nucleated cells including neutrophils. Instructions for isolating leukocytes (i.e. PBMCs and granulocytes) from whole blood samples can be found in the Isolation of Leukocytes, Bone Marrow and Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing Demonstrated Protocol.
6) Sorting neutrophils before sequencing may be desirable to improve the number of neutrophils assayed and reduce total sequencing costs. Sorted cells should be deposited directly into 1x PBS with 0.04% BSA (i.e. the recommended input buffer for 10x single-cell assays), and the sorted single-cell solution should be used as the 10x assay input. Forward and side scatter should be used to exclude cell doublets.
7) The 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 or RNA/cell and another with very little RNA/cell, the latter may be incorrectly labeled as background. Thus CellRanger may filter out the neutrophils which do not have a lot of mRNAs. We recommend running the analysis using the raw barcode matrix. Alternatively, the “force cell” parameter can be used with CellRanger to obtain cells with fewer total UMI counts.
As neutrophils exhibit the largest amount of retained introns compared to other cell types in peripheral blood samples (Wong et al, 2013, Ulrich & Guigo, 2020), the "Intron Mode" parameter in CellRanger(--include-introns) can be used to recover reads assigned to introns. By activating this parameter, intronic reads will be considered in the analysis, increasing the overall UMIs per barcode and thus, increasing neutrophil discovery.
We have successfully identified granulocytes in whole blood samples processed using the leukocyte isolation instructions in the Isolation of Leukocytes, Bone Marrow and Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing Demonstrated Protocol.
A few neutrophil-relevant 10x Genomics papers are listed below:
- https://www.biorxiv.org/content/10.1101/792200v1.full.pdf
- https://www.sciencedirect.com/science/article/pii/S1097276519301376
- https://science.sciencemag.org/content/360/6390/758
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969381/
- https://link.springer.com/content/pdf/10.1007/s13238-020-00752-4.pdf
Products: Single Cell Gene Expression, Single Cell Immune Profiling