Isolated nuclei from frozen tissue are the superior source for single cell RNA-seq compared with whole cells.

Abstract

The isolation of intact single cells from frozen tissue is a challenge due to the mechanical and physical stress inflicted upon the cell during the freeze-thaw process. Ruptured cells release ambient RNA into the cell suspension, which can become encapsulated into droplets during droplet based single cell RNA-seq library preparation methods. The presence of ambient RNA in droplets has been suggested to impact data quality, however there have been limited reports on single cell RNA-seq data from frozen tissue. Here, we compare the results of single cell RNA-seq derived from disaggregated cells from frozen brain tissue with single nuclei RNA-seq derived from purified nuclei of identical tissue using the 10X Genomics Chromium 3gene expression assay. Our results indicated that presence of ambient RNA in the cell suspension resulted in single cell RNA-seq data with a 25-fold lower gene count, a 5-fold lower UMI count per cell and a 2-fold lower fraction of reads per cell compared with single nuclei RNA-seq data. Cell clustering with the single cell RNA-seq data was unable to resolve the heterogeneity of brain cell types. Our conclusion is that nuclei from frozen tissue are the superior substrate for single cell transcriptome analysis.