title: GSM8193047: ID:D0071_Subject:73_Brain-Region:Precuneous (BA7)_Diagnosis:AD; Homo sapiens; RNA-Seq
library_construction_protocol: Fresh frozen brain tissue samples were thawed and micro-dissected, removing most white matter, and finely chopped using a blade. Tissue homogenization was performed in buffer containing 250mM sucrose and 0.1% TritonX100, using a glass Dounce with 0.02-0.055 mm clearance until no big clumps were visible (~30 strokes). The cellular nuclei fraction was purified using density gradient centrifugation, with the sample diluted in a 25% Iodixanol solution and layered on top of a 29% Iodixanol solution, then centrifuged at 13500g. Debris was carefully removed from the top layer and the nuclei pellet resuspended in PBS buffer containing 0.05~0.5% BSA and 2U/ml of DNAse I.  Immunostaining followed by FACS was used to remove nuclei aggregates using DAPI staining, and enrich for neuronal cells using NeuN. We used either a modified Drop-seq method or the standard 10x Genomics Chromium Single Cell 3' v2 or v3 assays. For Drop-seq, single nuclei were diluted to 200,000 cells/ml. To encapsulate individual nuclei and barcoded beads (Chemgenes, cat# Makosko-2011-10), we employed a microfluidic system (FlowJEM) and adjusted the flow parameters to generate ~100 µl (~0.5 nl) droplets (200 nuclei/µl; 165 beads/µl; flow rate: 3 ml/h). Lysis buffer contained 1% sarkosyl. Droplet-encapsulated nuclei were heated for 5 min at 72°C. Reverse transcription and PCR amplification followed previously described protocols (doi.org/10.1016/j.cell.2015.05.002). For 10x Genomics, ~12,500 nuclei were loaded to capture around 5,000 nuclei per sample. cDNA amplification and library construction followed the manufacturer's instructions.