Prayag Rajagopalan

Microglia are a unique type of glia in the central nervous system (CNS) that act as specialized macrophages in the central nervous system by maintaining neuronal connections, regulating brain development, and repairing injuries. Like other immune cells, microglia can exist in either a resting or activated state. They remain at rest in a healthy brain until they sense a threat, at which point they activate and dramatically change morphology and gene transcription. Their role in the CNS is thus crucial for healthy brain function, but little is known about their nuclear mechanism and function. One area of research delving into this subject is the cell-type specific temporal order of DNA replication, called Replication Timing (RT). During the S-phase of the cell cycle, the genome of a cell is replicated in a very specific order, whose discrete domains are named Replication Domains (RDs). Repli-Seq is a high-throughput sequencing method developed by Dr. Rivera-Mulia that segregates the S-phase of cells into separate early and late Replication Domains (RDs). In an attempt to learn more about the nuclear mechanisms of microglial cells in both states, I have optimized the protocol for culturing and activating microglial cells as well as obtaining data from these cells for Repli-Seq analysis. Our results showed that we needed a unique activation protocol to initiate activation for the cell line type we used. Furthermore, we optimized the nuclei processing and data analysis for both resting and activated microglial cell types to provide sufficient and high-quality data.