Core B: Molecular Biology Core

Project Summary/Abstract In the past few years, methods to determine the molecular identity neurons that have been labeled by the location of their projection outputs, the location of neurons providing specific synaptic input, or their ability to respond to a specific stimulus. Taking advantage of these approaches involves access to highly specialized equipment as well as the acquisition and application of expertise across myriad unrelated disciplines, including, but not limited to: microfluidics, low-volume pipetting, macromolecular crowding, bioanalyzer analysis, sequencing chemistry, large data set management, coding, generalized linear models, graph-based clustering, non-negative matrix factorization, multiple hypothesis testing correction, etc. Outside of the Molecular Biology Core, none of this program project grant laboratories has generated or analyzed NGS data, has significant experience with modern cloning approaches such as isothermal (Gibson) or Type IIS (Golden Gate) Assembly, or designed and used RNAScope probes. As each group is unlikely to perform such experiments with a frequency that would justify the time and expense necessary to develop those skills, centralization of these specialized techniques, increases consistency and quality and reduces cost/time waste resulting from duplicative/inefficient skills acquisition. In recruiting Dr. Linus Tsai to fill the role of director, the Molecular Biology Core leverages existing capabilities within his lab, consolidating support services for mouse husbandry and what are more expensive and/or technically challenging methods (RNAScope, cloning, bulk and single nucleus RNA-seq) to experienced laboratory and bioinformatic personnel. The Core’s experience with these methods maximizes the likelihood that these complex and difficult experiments are successful, and in turn that the aims outlined are achieved. In enabling PPG access to these state-of-the-art methods, we hope to provide PPG members with a basic understanding of how and when to use these techniques while demonstrate how molecular profiling can quickly facilitate the identification of pharmacotherapies that would be predicted to increase ventilatory response to hypercarbia.