Development Of Single-Cell Transcriptomic & Epigenomic Sequencing Technologies To Assess Cell-Type-Specific Gene Regulatory Programs In Mammalian Brains
Recent efforts to develop single-cell technologies have played a pivotal role in our ability to gain a high-resolution understanding of how tissue heterogeneity impacts human health, development, and disease. These technologies have become so powerful and popular in such a short period of time, and scientists and entrepreneurs alike are beginning to realize their potential. Concerted efforts are underway to generate “comprehensive reference maps of all human cells – the fundamental units of life – as a basis for both understanding human health and diagnosing, monitoring, and treating disease.” This goal belongs to The Human Cell Atlas, which is a world-wide consortium of scientists ready to advance on this truly massive undertaking to map all aspects of human health, diversity, and wellness at the single cell level. Measurements of single-cell genomes, transcriptomes, epigenomes (methylomes/genome architecture) at all stages and states of human existence will be included, and to this end, new technologies are constantly being developed and refined to contribute to this venture. In this thesis, I will discuss two single-cell technologies that I developed, which will deepen our understanding of how cells function. Both of our techniques are built upon the current state-of-the art in single cell technologies to address major limitations of current techniques and contribute to the powerful repertoire of single-cell transcriptomics/epigenomics. First, we identified significant challenges associated with single-cell isolation and dissociation from complex adult tissues. Chemical or enzymatic dissociation of complex tissues elicits stress response pathways and ectopic transcription, confounding single-cell RNA-seq data. In addition, cells from previously frozen tissues have compromised cellular membranes, which limits our ability to generate high-quality single-cell isolates. We identified that single-nucleus input might address these pitfalls associated with whole-cell isolation, because nuclei can be mechanically isolated from complex adult tissues and are resistant to the freeze-thaw process. Thus, we set out to develop a single-nucleus RNA sequencing strategy using the Drop-seq microfluidic platform. To this end, we successfully developed sNucDrop-seq which illustrated that single-nucleus RNA libraries could be utilized to reflect cellular heterogeneity and were also primed to assay activity-dependent transcriptional dynamics due to the primarily nascent nature of transcripts localized in the nucleus. In addition, we were the first to develop a base-resolution single-cell 5-hydroxymethylome (5hmC)- and “true” 5-methylcytosine (5mC) joint-profiling technology to assess the relationship of two DNA modifications with transcriptional outcomes in the same single cell. The current state-of-the-art DNA methylome profiling technologies rely solely on bisulfite conversion, which cannot distinguish 5hmC from 5mC. Because 5hmC is significantly enriched and seemingly stable in the central nervous system (CNS), and 5hmC and 5mC are associated with opposing transcriptional outcomes, it is essential to resolve this base-ambiguity to fully understand epigenetic regulatory mechanisms in 5hmC-enriched cells.