NextSeq 500 paired end sequencing; Single-cell sequencing of cerebral organoids with familial Alzheimer's disease (AD) mutation in PSEN1 and non-demented control
Single-cell sequencing of cerebral organoids with familial Alzheimer's disease (AD) mutation in PSEN1 and non-demented control
For each scRNA-seq sample, 18 COs were used. Whole organoids were washed in HBSS, cut into pieces and placed into collagenase P dissolved in HBSS at 0.5 mg/ml. Dissociation was performed in a shaking incubator for 15 min at 37 °C with occasional pipetting, followed by ten-fold dilution with cold 2% FBS in HBSS and filtering through 40 µm cell strainer. Cells were centrifuged and resuspended in 500 μl of 2% FBS in HBSS. The cell suspension was stained for viability with 7-AAD, and 500,000 viable cells were sorted. Single-cell partitioning and cDNA preparation were done using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics) according to the manufacturer's protocol Sequencing library preparation was done according to the manufacturer's protocol (Chromium Single Cell 3' Reagent Kits User Guide v3.1).