Progress in Assessing Retinal Microglia Using Single-Cell RNA Sequencing

Retinal degeneration is the leading cause of both inherited and age-related vision loss, and often leads to neuroimmune activation and a heterogeneous population of microglia and monocyte-derived macrophages. A common method to study the innate immune response during retinal degeneration is single-cell RNA sequencing, but the best way to obtain and analyze these cells over the course of degeneration remains debated. Here, we compare two common methods of retinal cell preparation (collagenase digestion with immune cell enrichment by FACS; Col/FACS vs papain digestion of the whole retina without enrichment; Pap/Whole) and three different algorithms for database integration (CCA, RPCA, and Harmony) to examine microglia in healthy retinas. We find that the Pap/Whole dissociation produced a smaller fraction of activated microglial cells and that the Harmony integration of microglia isolated by these two methods resulted in the highest Silhouette score, indicating the greatest separation of microglia subclusters from these data sets.