GSM6185796: snRNAseq_LaFet1; Homo sapiens; RNA-Seq
25mg of frozen postmortem human brain tissue was homogenized in cold lysis buffer (0.32M Sucrose, 5 mM CaCl2, 3 mM Magnesium acetate, 0.1 mM, EDTA, 10mM Tris-HCl, pH8, 1 mM DTT, 0.1% Triton X-100) and filtered through a 40 µm cell strainer. 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 250 µl with PBS and 7AAD (Invitrogen) was added according to manufacturer's instructions. 7AAD positive nuclei were sorted into tubes pre-coated with 5% BSA using a FACSAria flow cytometer (BD Biosciences). Nuclei were subjected to 2 washes in 200 µl wash buffer (10x Genomics), after which they were re-suspended in 30 µl nuclei buffer (10x Genomics) and quantified (Countess II, Life Technologies). 8000 nuclei from each sample were subjected to the Chromium Next GEM Single Cell Multiome ATAC-seq and Gene Expression protocol (10x Genomics), according to manufacturer's instructions. Resulting libraries were quantified using the KAPA library quantification kit (KAPA Biosystems) and fragment sizes determined by Tapestation (Agilent). All libraries were sequenced at New York Genome Center (NYGC) using the Novaseq platform (Illumina).