Novaseq 6000 s2 sequencing kits

We isolated nuclei from frozen postmortem brain tissue as previously described (Mathys et al., Nature 2019)^{65 (link)} with some modifications. Briefly, we homogenized the brain tissue in 700 μL Homogenization buffer and filtered the homogenate through a 40 μm cell strainer (Corning, NY), added 450 μL Working solution and loaded it as a 25% OptiPrep solution on top of a 30%/40% OptiPrep density gradient (750 μL 30% OptiPrep solution, 300 μL 40% OptiPrep solution). We separated the nuclei by centrifugation using a fixed rotor, tabletop centrifuge (5 minutes, 10000g, 4°C). We collected the nuclei pellet at the 30%/40% interphase, transferred it on a new tube, washed it twice with 1mL ice-cold PBS containing 0.04% BSA (centrifuged 3 minutes, 300g, 4°C) and finally resuspended it in 100 μL PBS containing 0.04% BSA. After counting, we diluted the nuclei to a concentration of 1000 nuclei per μL. We used the isolated nuclei for the droplet-based 10x scRNA-seq assay, targeting 5000 nuclei per brain region and individual, and prepared libraries using the Chromium Single-Cell 3′ Reagent Kits v3 (10x Genomics, Pleasanton, CA) according to the manufacturer’s protocol. We sequenced pooled libraries using the NovaSeq 6000 S2 sequencing kits (100 cycles, Illumina).