Hi! Could someone kindly clarify to me how the Seattle Alzheimer's Disease Brain Cell Atlas (SEA-AD) Study data was generated (syn26223298). I'd specifically like to know whether brain nuclei were sorted based on protein markers before sequencing for this dataset and the MTG reference dataset. If nuclei were sorted, were they then re-pooled at specified proportions and loaded into a single well on the 10x micro-fluidic chip per donor brain? Or was it that each subset of sorted nuclei was loaded on a separate well without being re-pooled. The reason I'm asking is that I read the documentation about SEA-AD and MTG reference data generation on this website: https://portal.brain-map.org/explore/seattle-alzheimers-disease/seattle-alzheimers-disease-brain-cell-atlas-download?edit&language=en And it appears to me like nuclei were only sorted based on the neuronal marker NeuN. Yet, the sequencing data for each inferred cell type is provided discretely here as if multiple markers were used to sort nuclei then individually perform library preparation and sequencing on each set of sorted nuclei: https://cellxgene.cziscience.com/collections/1ca90a2d-2943-483d-b678-b809bf464c30 Thank you for your assistance.

Created by Rached Alkallas ralkallas
Hello, Nuclei were sorted based on DAPI and NeuN fluorescence intensity. Nuclei were excluded from debris based on DAPI signal and were gated to exclude multiplets. Nuclei were then gated on NeuN fluorescence intensity and NeuN+ and NeuN- nuclei were sorted into separate FACS tubes. Sorted NeuN+ and NeuN- were then pooled together at a defined ratio (70% NeuN+, 30% NeuN-) and the pooled sample was loaded on a single well of a 10x micro-fluidic chip. I hope this is helpful to you. All the best, Eitan S. Kaplan, PhD
@eitan.kaplan Can you help answer this question?

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