Question: Can I process neutrophils using 10x Single Cell applications?
Answer: Neutrophils are very low in RNA content compared to other blood cell types. In addition, they can be rich in RNases; which potentially inhibit the reaction, resulting in fewer transcripts detected in GEMs and less usable sequencing reads.
A few points to consider when optimizing the Single Cell Assays for your particular sample type:
1) Additional wash steps can help remove some of the RNase 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)
2) 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.
3) Increasing the PCR cycles by 2 extra cycles during cDNA amplification in a non-enriched sample. This may help to bring the neutrophil-specific transcripts above background.
4) 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. There is an easy way to manually adjust the cutoff based on the ranked barcode plot, which is one of our QC methods to assess the quality of your data and sample.
A few neutrophil-relevant 10x papers are listed below:
Products: Single Cell Gene Expression, Single Cell Immune Profiling