title: GSM5421001: SPMS [S2]; Homo sapiens; RNA-Seq
library_construction_protocol: Post-mortem prefrontal cortices (BA10) from donors who suffered from RRMS or SPMS were cryo-sectioned at 300 µm. The buffers used in this procedure were made in autoclaved diethyl pyrocarbonate (DEPC; MilliporeSigma, Cat# D5758)-treated water. All the steps from here on were performed at 4 ºC with additional equilibration time at each step. The samples were initially placed in 0.5 mL of nuclear extraction buffer (NEB; 0.32 M Sucrose, 5 mM CaCl2.2H20, 3 mM (CH3COO)2Mg.4H2O, 0.1 mM EDTA, 10 mM Tris/HCl (pH 8.0), 0.1% Triton X-100 with cOmpleteTM mini protease inhibitor cocktail tablet (MilliporeSigma, Cat# 11836153001) and fresh 0.2% RNase inhibitor (Takara, Cat# 2313A)) for 15 minutes. After adding another 0.5 mL NEB, the samples were homogenized thoroughly with a Dounce homogenizer. Upon transferring this homogenate to a new tube, the Dounce homogenizer was rinsed with 1 mL of NEB. Homogenates were then passed through a CellTrics® 50 µm filer (Sysmex, Cat# 04-004-2324). After washing the filter with NEB once, the filtrates were centrifuged at 1600 x g for 5 mins. The resulting pellets were resuspended with 1 mL of PBSE (1X PBS, 2 mM EGTA and fresh 0.2% RNase inhibitor), transferred into a new tube, diluted with PBSE, and centrifuged again at 1600 x g for 5 mins. Meanwhile, density gradients of OptiPrepTM (MilliporeSigma, Cat# D1556) were made in FACS tubes with 0.4 mL 35% OptiPrepTM on bottom and 2 mL 10% OptiPrepTM on top. Solutions used for density gradients were as follows: OptiPrepTM, solution B (6X concentrations: 120 mM Tricine/NaOH pH 7.8, 150 mM KCl and 30 mM MgCl2.6H2O) and solution D (0.25 M Sucrose in 1X solution B with freshly added 0.2% RNase inhibitor). The OptiPrepTM solutions were diluted with solution D to make the final concentrations to 35% and 10%. Nuclear pellets were resuspended with 0.2 mL solution D, followed by addition of 0.2 mL of 10% OptiPrepTM solutions, which were layered onto the 10% OptiPrepTM solution, and centrifuged at 3250 x g for 10 mins. The nuclei that settled at the boundary of 35% and 10% were collected, washed twice with PBSE containing 1% fatty acid-free bovine serum albumin (BSA; Gemini Bio, Cat# 700-107P), passed through CellTrics® 50 µm filter, washed again with PBSE + BSA, and labeled with DAPI (1:100,000, 4',6-diamidino-2-phenylindole; MilliporeSigma, Cat# D9542). About 3 x 105 nuclei were sorted using a BD FACSAria™ II (BD Biosciences) into a precoated collection tube filled with 1 mL of PBSE + BSA. The nuclei were washed once and resuspended in PBSE + BSA to obtain around 1500 cells/µL, and the counts were confirmed using a CountessTM II (Thermo Fisher Scientific). Single-nucleus capture (target capture of 5,000 nuclei per sample) and library preparation was conducted using Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3 (10x Genomics, Cat# PN-1000075) and Chromium Single Cell B Chip Kit (10X Genomics, Cat# PN-1000074) according to the manufacturer's instruction. Single-nucleus libraries were prepared using standard Illumina protocols and were sequenced on the Illumina NovaSeq 6000 machine at GENEWIZ, Inc.