- Preparing Highly Viable Single-Cell Suspensions from Mouse Pancreatic Islets for Single-Cell RNA Sequencing.
Preparing Highly Viable Single-Cell Suspensions from Mouse Pancreatic Islets for Single-Cell RNA Sequencing.
STAR protocols (2020-12-31)
Hugo Lee, Feyza Engin
PMID33377038
ABSTRACT
Pancreatic islets consist of several cell types, including alpha, beta, delta, epsilon, and PP cells. Due to cellular heterogeneity, it is challenging to interpret whole-islet transcriptome data. Single-cell transcriptomics offers a powerful method for investigating gene expression at the single-cell level and identifying cellular heterogeneity and subpopulations. Here, we describe a protocol for mouse pancreatic islet isolation, culturing, and dissociation into a single-cell suspension. This protocol yields highly viable cells for successful library preparation and single-cell RNA sequencing. For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).
MATERIALS
Product Number
Brand
Product Description
Sigma-Aldrich
Bovine Serum Albumin, fatty acid free, low endotoxin, lyophilized powder, BioReagent, suitable for cell culture, ≥96% (agarose gel electrophoresis)
Sigma-Aldrich
Collagenase from Clostridium histolyticum, Type XI, 2-5 FALGPA units/mg solid, ≥800 CDU/mg solid
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture
Kimwipes® disposable wipers, L × W 4 1/2 in. × 8 1/2 in.