Single-nucleus chromatin profiling of aged non-PD control
Human midbrain samples were obtained through NIH NeuroBioBank requests (The Human Brain and Spinal Fluid Resource Center, University of Maryland Brain and Tissue Bank, Harvard Brain Tissue Resource Center, University of Miami Brain Endowment Bank).   To reduce batch effects, tissue blocks were processed in a random order, with each batch containing at least 1 of each donor type.  Two batches of four tissue blocks were processed each day until all donor samples had been processed.  To capture the SN region of the midbrain precisely, we dissected approximately 3 x 3 x 5 mm sections using postmortem photos provided (Supplementary Fig.1a), yielding tissue blocks ranging from 50-75 mg. We processed the tissue in a dounce homogenizer and then centrifuged it through 1.5 M sucrose to isolate the nuclei. With this method, we were able to reliably isolate > 1x106 intact nuclei from ~50 mg postmortem midbrain tissue. Centrifugation through an iodixanol gradient as previously described9 produced high-quality nuclei with low mitochondrial contamination, but the actual yield of nuclei was very low (2.5-5x104 nuclei from ~50 mg postmortem midbrain tissue), so we did not continue with this method as the SN is a small region and tissue amount is limited. We stained the nuclei with 7AAD and sorted to purify nuclei from mitochondria. After sorting, we permeabilized according to current 10XGenomics protocols (Protocol CG000375 Rev A: 10 mMTris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.01% Digitonin, 1% BSA, 1 mM DTT, 1 U/uL RNase Inhibitor) for 2 min on ice).  Nuclei were washed once before counting and proceeding with 10XGenomics protocols for transposition, nuclei isolation and barcoding, and library preparation exactly as written (10XGenomics Protocol Chromium Next GEM Single Cell Multiome ATAC + Gene Expression CG000338 Rev A). Libraries were sequenced at GeneWiz using Illumina Novaseq S4 flowcells.  Our actual average sequencing depth was 1.8E8 reads/sample for gene expression libraries and 1.75E8 reads/sample for ATAC libraries. After sequencing, three samples (one each of Young, Aged, and PD) showed strong ATAC results but poor RNA read quality indicative of RNA degradation in the original sample. The nuclei from these three donors were excluded from downstream analysis.  