Single Cell Transcriptome Analysis of Peripheral Blood Mononuclear Cells in Freshly Isolated versus Stored Blood Samples

Abstract

Background: Peripheral blood mononuclear cells (PBMCs) are widely used as a model in the study of different human diseases. There is often a time delay from blood collection to PBMC isolation during the sampling process, which can result in an experimental bias, particularly when performing single cell RNA-seq (scRNAseq) studies. Methods: This study examined the impact of different time periods from blood draw to PBMC isolation on the subsequent transcriptome profiling of different cell types in PBMCs by scRNAseq using the 10X Chromium Single Cell Gene Expression assay. Results: Examining the five major cell types constituting the PBMC cell population, i.e., CD4+ T cells, CD8+ T cells, NK cells, monocytes, and B cells, both common changes and cell-type-specific changes were observed in the single cell transcriptome profiling over time. In particular, the upregulation of genes regulated by NF-kB in response to TNF was observed in all five cell types. Significant changes in key genes involved in AP-1 signaling were also observed. RBC contamination was a major issue in stored blood, whereas RBC adherence had no direct impact on the cell transcriptome. Conclusions: Significant transcriptome changes were observed across different PBMC cell types as a factor of time from blood draw to PBMC isolation and as a consequence of blood storage. This should be kept in mind when interpreting experimental results.

Keywords

• AP-1
• NF-kB
• peripheral blood mononuclear cells
• single cell RNA-seq