183 fresh-frozen post-mortem caudate brain samples were divided into 23 pools, with 8 in each pool. The donors in each pool were both condition (with or without AUD) and sex balanced. For each pool, around 20 mg tissue from each donor specimen was collected and combined into a sterilized 2 ml Dounce homogenizer. 2 ml chilled NP40 lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet P40 Substitute, 1 mM DTT, 1 U/µl RNase inhibitor) was added to the Dounce homogenizer before the tissues were thawed. The tissues were homogenized 15× using pestle A, and 10× using pestle B, and were transferred into a centrifuge tube to incubate for 2 min on ice. After that, 2 ml wash buffer containing PBS, 1% BSA, and 1 U/µl RNase inhibitor was added and mixed well. The lysed tissue was centrifuged at 500 × g for 5 min at 4 °C, then washed twice more with wash buffer and filtered through 70 µm and then 40 µm cell strainer separately. The pellet was resuspended in 2 ml
