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Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing.
Foster, Deshka S; Griffin, Michelle; Januszyk, Michael; Delitto, Daniel; Norton, Jeffrey A; Longaker, Michael T.
Affiliation
  • Foster DS; Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Stanford University.
  • Griffin M; Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Stanford University.
  • Januszyk M; Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Stanford University.
  • Delitto D; Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Stanford University.
  • Norton JA; Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Stanford University.
  • Longaker MT; Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Stanford University; longaker@stanford.edu.
J Vis Exp ; (200)2023 10 13.
Article in En | MEDLINE | ID: mdl-37902368
ABSTRACT
Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.
Subject(s)

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Cell Nucleus / Neoplasms Limits: Humans Language: En Journal: J Vis Exp Year: 2023 Document type: Article

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Cell Nucleus / Neoplasms Limits: Humans Language: En Journal: J Vis Exp Year: 2023 Document type: Article