MicroC of sorted nuclei from pooled MTG nuclei
To obtain sufficient nuclei for Micro-C, we utilized middle temporal gyrus tissue samples from 36 individuals with PD, specifically with neocortical Lewy body pathology, and 21 healthy controls. Because microglia and astrocyte nuclei represent a small minority of the total nuclei recovered by Dounce homogenization, we pooled nuclei isolated from dozens of donors prior to sorting. In this pooling strategy, PD and control individuals were kept separate, but we did not attempt to uncover differences between the Micro-C signal of these groups. After isolating nuclei in the same manner as previously mentioned, nuclei were cryopreserved for later pooling and nuclei sorting. After combining sufficient nuclei, we employed fluorescence-activated nuclei sorting to obtain the 4 major brain cell types: microglia, astrocytes, oligodendrocytes, and neurons. Due to the low percentage yield of microglia and astrocytes from this process, the samples were split into 2 batches based on disease state with ~150M nuclei each and sorted simultaneously using two BD FACS Aria Fusion machines. In this pooling process, the batches were optimized to balance the number of nuclei per individual to prevent bias toward any one donor. Biological and technical replicates for each cell type were then processed via Dovetails Micro-C Kit.