Cellular dissociation and transcriptome analysis of adult mouse cortex
Despite strong evidence of a genetic contribution to many complex diseases, such as Alzheimer’s disease, the underlying genetic mechanism remains elusive. This problem may be explained as a natural consequence of the deep complexity of cell specialization. Transcriptome analysis of a tissue homogenate is incapable of detecting the abnormalities in an individual cell. In this study, we developed methods for isolation of single cells from mouse cortex performed with single cell 3’ RNA sequencing to identify individual cell types present in the tissue. Single cell suspensions were prepared by two methods, the Miltenyi gentleMACS system and mechanical dissociation with a Dounce homogenizer. Both methods were followed by a debris removal step via gradient centrifugation to reduce background noise. The mRNA of each cell was barcoded using the 10X Chromium 3’ single cell system, and the resulting cDNA libraries were sequenced on the Illumina MiSeq platform. Reads were aligned to the mouse (Mm10) transcriptome reference and gene counts for each cell were computed. Cell types were assigned to each cell based on a consensus of marker gene counts. Dounce homogenization produced cell suspensions with little background contamination as assessed by NucBlue nuclear stain and Cellmask membrane stain. The gentleMACS system produced cell suspensions with greater background debris based on microscopy. However, the background noise was less severe in the gentleMACS suspension based on the final sequencing results. The classifiers were capable of differentiating between various cell types, although only three cell types were identified. This demonstrates that background debris assessed by microscopy is unreliable. The two methods juxtaposed in our experiment provided unique cell types, and although the mechanical isolation of the Dounce homogenizer did produce more single cells, the results revealed that both procedures have distinct strengths and weaknesses that need to be addressed further.