Nuclei were isolated from 24 biospecimens of frozen human postmortem brain tissue (12 individuals (Unaffected/OUD) x 2 brain regions = 24 samples). Samples weighing 10–15 mg were homogenized using ~10 strokes per glass pestle in 7 mL glass douncers with 5 mL nuclei isolation medium containing DAPI. Homogenate was filtered using a 40um mesh strainer (Fisher Scientific #48680). Nuclei were sorted for DAPI fluorescence using a BD FACS Aria at the Boston University Flow Cytometry Core. Approximately 100,000 nuclei were sorted into 7ul of 0.04% bovine serum albumin (Millipore Sigma #126615) in phosphate buffered saline (ThermoFisher #10010031). Nuclei were counted using a hemocytometer and assessed for concentration and debris. 7000 nuclei were targeted per sample except for one sample with lower concentration where 5000 nuclei were targeted. The 10x Chromium process was performed and next generation sequencing libraries were prepared using the 10x genomics single cell 3’ gene expression dual index kit.
