RNA-sequencing From Single Nuclei

It has recently been established that double-stranded cDNA can be synthesized from the polyadenylated RNA of a single cell in quantities sufficient for analysis of global gene expression using DNA sequencing. From the number of sequence reads mapping to each gene, transcript levels are quantified, and information about mutations and mRNA splicing variants emerges from the individual sequence reads. Here we demonstrate that this approach can be done using the extremely low levels of mRNA found within a single nucleus, isolated either from a mouse neural progenitor cell line, or from dissected hippocampal tissue. As implemented with single nuclei, this method provides excellent coverage and technical reproducibility. On average, expression of more than 16,000 of the 24,057 mouse protein-coding genes was detected in each nucleus, and the degree of gene-expression variation was similar when single nuclei and single cells were compared. Major advantages of this method include: first, nuclei, as compared to whole cells, can be easily isolated from complex tissues and organs. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. Third, it also provides insight into regulatory mechanisms specific to the nucleus. Fourth, the method enables dissection of regulatory events at the single-cell level, which should reveal the physiological state and interconnectedness of gene regulation that are masked when studied by conventional transcriptomics using bulked cells or tissues.