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  • 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
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
Collagenase from Clostridium histolyticum, Type XI, 2-5 FALGPA units/mg solid, ≥800 CDU/mg solid
Pricing and availability is not currently available.
Sigma-Aldrich
Bovine Serum Albumin, fatty acid free, low endotoxin, lyophilized powder, BioReagent, suitable for cell culture, ≥96% (agarose gel electrophoresis)
Pricing and availability is not currently available.
Sigma-Aldrich
Histopaque®-1077, sterile-filtered, density: 1.077 g/mL
Pricing and availability is not currently available.
Sigma-Aldrich
Dulbecco′s Phosphate Buffered Saline, Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell culture
Pricing and availability is not currently available.
Kimwipes® disposable wipers, L × W 4 1/2 in. × 8 1/2 in.
Pricing and availability is not currently available.