GSM7108115
Isolation and fluorescence-activated nuclear sorting (FANS) of nuclei for frozen human postmortem brain:  Samples were processed in batches consisting of 2 individuals; 1 case and 1 control. For each case, we processed 3 dissections; 1 of gray matter, 1 white matter, and 1 containing an MS plaque. For each Control we processed 2 dissections; 1 gray matter and 1 white matter. Approximately 25 mg of frozen postmortem human brain tissue from each dissection was homogenized in cold lysis buffer (0.32M Sucrose, 5 mM CaCl2, 3 mM Magnesium acetate, 0.1 mM, EDTA, 10 mM Tris-HCl, pH8, 1 mM DTT, 0.1% Triton X-100) and filtered through a 40 µm cell strainer. All buffers were supplemented with RNAse inhibitors (Takara). The flow-through was underlaid with sucrose solution (1.8 M Sucrose, 3 mM Magnesium acetate, 1 mM DTT, 10 mM Tris-HCl, pH8) and centrifuged at 107,000 g for 1 hour at 4°C. Pellets were resuspended in PBS supplemented with 0.5% bovine serum albumin (BSA). Prior to FANS, volumes were brought up to 500 µl with PBS and DAPI (Thermoscientific) added to a final concentration of 1 µg/ml. DAPI-positive nuclei were sorted into tubes pre-coated with 5% BSA using a FACSAria flow cytometer (BD Biosciences). Single-nuclei gene expression library preparation:  Following FANS, nuclei were quantified (Countess II, Life Technologies) and 8,000 from each sample loaded on a single 10x lane using single cell 3' v3 reagents (10x Genomics). cDNA libraries were prepared according to the manufacturer's protocol. Libraries were sequenced at New York Genome Center using the Novaseq platform (Illumina). snRNA-seq