Name: single-cell rna sequencing and analysis of human pancreatic islets
Uploaded: 2019-07-18T14:00:00
Description: Watch this Scientific Journal Video about Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets at JoVE.com

1. Human islet dissociation
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Place the islets in a bottle and immerse it completely in the islet medi...

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...

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: &#945;-cells produce glucagon, &#946;-cells insulin, &#948;-cells somatostatin, PP cells pancreatic polypeptide, and &#949;-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 sequencing2,3,4,5,6,7,8. Although the whole islet transcriptome is informative to identify the organ-specific transcripts and disease candidate genes, it fails to uncover the molecular heterogeneity of each islet cell type. Laser capture microdissection (LCM) technique has been applied to directly obtain specific cell types from islets9,10,11,12 but falls short of purity of the targeted cell population. To overcome these limitations, fluorescence-activated cell sorting (FACS) has been used to select specific endocrine cell populations, such as &#945;- and &#946;-cells13,14,15,16,17,18. Moreover, Dorrell et al. used an antibody-based FACS sorting approach to classify &#946;-cells into four subpopulations19. FACS-sorted islet cells can also be plated for RNA sequencing of single cells; however, the plate-based methods face challenges in scalability20,21,22. 
To generate high quality, large-scale transcriptome data of each endocrine cell type, we applied microfluidic technology to human islet cells. The microfluidic platform generates transcriptome data from a large number of single cells in a high-throughput, high-quality, and scalable manner23,24,25,26,27. In addition to revealing molecular characteristics of a cell type captured in a large quantity, highly-scalable microfluidic platform enables identification of rare cell types when enough cells are provided. Hence, application of the platform to human pancreatic islets allowed profiling of ghrelin-secreting &#949;-cells, a rare endocrine cell type with little known function due to its scarcity28. In recent years, several studies have been published by us and others reporting large-scale transcriptome data of human islets using the technology29,30,31,32,33. The data are publicly available and useful resources for the islet community to study endocrine cell heterogeneity and its implication in diseases. 
Here, we describe a droplet-based microfluidic single-cell RNA sequencing protocol, which has been used to produce transcriptome data of approximately 20,000 human islet cells including &#945;-, &#946;-, &#948;-, PP, &#949;-cells, and a smaller proportion of non-endocrine cells32. The workflow starts with isolated human islets and depicts steps of islet cell dissociation, single-cell capture, and data analysis. The protocol requires the use of freshly isolated islets and can be applied to islets from humans and other species, such as rodents. Using this workflow, unbiased and comprehensive islet cell atlas under baseline and other conditions can be built.

<table>
	<tbody>
		<tr>
			<td>30 &#181;m Pre-Separation Filters</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td>Cell strainer</td>
		</tr>
		<tr>
			<td>8-chamber slides</td>
			<td>Chemometec</td>
			<td>102673-680</td>
			<td>Dell counting assay slides</td>
		</tr>
		<tr>
			<td>Bioanalyzer High Sensitivity DNA Kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td>for QC</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td>Single cell media</td>
		</tr>
		<tr>
			<td>Chromium Single Cell 3&#39; Library &#38; Gel Bead Kit v2, 16 rxns</td>
			<td>10X Genomics</td>
			<td>120237</td>
			<td>Single cell reagents</td>
		</tr>
		<tr>
			<td>Chromium Single Cell A Chip Kit v2, 48 rx (6 chips)</td>
			<td>10X Genomics</td>
			<td>120236</td>
			<td>Microfluidic chips</td>
		</tr>
		<tr>
			<td>CMRL-1066</td>
			<td>ThermoFisher</td>
			<td>11530-037</td>
			<td>Complete islet media</td>
		</tr>
		<tr>
			<td>EB Buffer</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td>Elution buffer</td>
		</tr>
		<tr>
			<td>Eppendorf twin-tec PCR plate, 96-well, blue, semi-skirted</td>
			<td>VWR</td>
			<td>47744-112</td>
			<td>Emulsion plate</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher</td>
			<td>16000-036</td>
			<td>Complete islet media</td>
		</tr>
		<tr>
			<td>Human islets</td>
			<td>Prodo Labs</td>
			<td>HIR</td>
			<td>Isolated human islets</td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM)</td>
			<td>ThermoFisher</td>
			<td>25030-081</td>
			<td>Complete islet media</td>
		</tr>
		<tr>
			<td>Nextera DNA Library Preparation Kit (96 samples)</td>
			<td>Illumina</td>
			<td>FC-121-1031</td>
			<td>Library preparation reagents</td>
		</tr>
		<tr>
			<td>NextSeq 500/550 High Output Kit v2.5 (75 cycles)</td>
			<td>Illumina</td>
			<td>FC-404-2005</td>
			<td>Sequencing</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher</td>
			<td>15140-122</td>
			<td>Complete islet media</td>
		</tr>
		<tr>
			<td>Qubit High Sensitivity dsDNA Kit</td>
			<td>Life Technologies</td>
			<td>Q32854</td>
			<td>for QC</td>
		</tr>
		<tr>
			<td>Solution 18</td>
			<td>Chemometec</td>
			<td>103011-420</td>
			<td>Cell counting assay reagent</td>
		</tr>
		<tr>
			<td>SPRISelect Reagent</td>
			<td>Fisher Scientific</td>
			<td>B23318</td>
			<td>Purification beads</td>
		</tr>
		<tr>
			<td>Tissue Culture Dishes (10 cm)</td>
			<td>VWR</td>
			<td>10861-594</td>
			<td>for islet culture</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Life Technologies</td>
			<td>12604-013</td>
			<td>Cell dissociation solution</td>
		</tr>
		<tr>
			<td>Zymo DNA Clean &#38; Concentrator-5, 50 reactions</td>
			<td>VWR</td>
			<td>77001-152</td>
			<td>Library clean up columns</td>
		</tr>
	</tbody>
</table>

single-cell rna sequencing and analysis of human pancreatic islets

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.

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 ...

Watch this Scientific Journal Video about Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets at JoVE.com

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

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.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

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