Preparation of single-cell suspension from mouse breast cancer focusing on preservation of original cell state information and cell type composition

Abstract

Single-cell analysis of gene expression has become a very popular method during the last decade. Unfortunately, appropriate standardization and workflow optimization remain elusive. The first step of the single cell analysis requires that the solid tissue be disassociated into a suspension of individual cells. However, during this step several technical bias can arise which can later result in the misinterpretation of the data. The goal of this study was to identify and quantify the effect of these technical factors on the quality of the single-cell suspension and the subsequent interpretation of the produced expression data. We tested the effects of various enzymes used for dissociation, several centrifugation forces, dissociation temperatures and the addition of Actinomycin D, a gene expression inhibitor. RT-qPCR was used to assess the effect from each parameter alteration, while a single-cell RNA sequencing experiment was used to confirm the optimized factors. Our concluding results provide a complete protocol for the tissue dissociation of mouse mammary tumour from 4T1 cells that preserves the original cell state and is suitable for any single-cell RNA sequencing analysis. Furthermore, our workflow may serve as a guide for the optimization of the dissociation procedure of any other tissue of interest, which would ultimately improve the reproducibility of the reported data.