Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Summary

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Abstract

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Introduction

Pancreatic islets release endocrine hormones to regulate blood glucose levels. Five endocrine cell types, which differ functionally and morphologically, are involved in this essential role: α-cells produce glucagon, β-cells insulin, δ-cells somatostatin, PP cells pancreatic polypeptide, and ε-cells ghrelin¹. Gene expression profiling is a useful approach to characterize the endocrine cells in normal or specific conditions. Historically, the whole islet gene expression profiling was generated using microarray and next-generation RNA sequencing^2,3,

Protocol

1. Human islet dissociation

1. Obtain human islets isolated from cadaver organ donors of either sex, ages between 15-80 years, without pre-existing diseases unless islets from donors with specific demographics are required for the study purpose.
   1. After isolation, have the isolated islets kept in the tissue culture facility for 2-3 days at the supplier. It often takes more than 1 day for islet damages to become visible.
   2. Place the islets in a bottle and immerse it completely in the islet medium. Get it to the laboratory by overnight shipment.
   3. Obtain the islet equivalent quantity (IEQ) of the shipped islets from the islet supplie....

Results

The single-cell RNA sequencing workflow consists of three steps: dissociating intact human islets into single cell suspension, capturing single cells using a droplet-based technology, and analyzing RNA-seq data (Figure 1). Firstly, the acquired human islets were incubated overnight. The intact islets were examined under the microscope (Figure 2A). The integrity of dissociated islet cells has been validated using RNA fluorescence .......

Discussion

Single-cell technologies developed in recent years provide a new platform to characterize cell types and study molecular heterogeneity in human pancreatic islets. We adopted a protocol of droplet-based microfluidic single-cell isolation and data analysis to study human islets. Our protocol successfully produced RNA sequencing data from over 20,000 single human islet cells with relatively small variations in sequence quality and batch effects.

In particular, two steps are critical in this proto.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
30 µm Pre-Separation Filters	Miltenyi Biotec	130-041-407	Cell strainer
8-chamber slides	Chemometec	102673-680	Dell counting assay slides
Bioanalyzer High Sensitivity DNA Kit	Agilent	5067-4626	for QC
Bovine Serum Albumin	Sigma-Aldrich	A9647	Single cell media
Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns	10X Genomics	120237	Single cell reagents
Chromium Single Cell A Chip Kit v2, 48 rx (6 chips)	10X Genomics	120236	Microfluidic chips
CMRL-1066	ThermoFisher	11530-037	Complete islet media
EB Buffer	Qiagen	19086	Elution buffer
Eppendorf twin-tec PCR plate, 96-well, blue, semi-skirted	VWR	47744-112	Emulsion plate
Fetal Bovine Serum	ThermoFisher	16000-036	Complete islet media
Human islets	Prodo Labs	HIR	Isolated human islets
L-Glutamine (200 mM)	ThermoFisher	25030-081	Complete islet media
Nextera DNA Library Preparation Kit (96 samples)	Illumina	FC-121-1031	Library preparation reagents
NextSeq 500/550 High Output Kit v2.5 (75 cycles)	Illumina	FC-404-2005	Sequencing
Penicillin-Streptomycin (10,000 U/mL)	ThermoFisher	15140-122	Complete islet media
Qubit High Sensitivity dsDNA Kit	Life Technologies	Q32854	for QC
Solution 18	Chemometec	103011-420	Cell counting assay reagent
SPRISelect Reagent	Fisher Scientific	B23318	Purification beads
Tissue Culture Dishes (10 cm)	VWR	10861-594	for islet culture
TrypLE Express	Life Technologies	12604-013	Cell dissociation solution
Zymo DNA Clean & Concentrator-5, 50 reactions	VWR	77001-152	Library clean up columns