Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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^{2,3,4,5,6,7,8}. 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 islets^9,10,11,12 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 α- and β-cells^{13,14,15,16,17,18}. Moreover, Dorrell et al. used an antibody-based FACS sorting approach to classify β-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 scalability^20,21,22.

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 manner^{23,24,25,26,27}. 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 ε-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 technology^{29,30,31,32,33}. 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 α-, β-, δ-, PP, ε-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.

Protokół

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 supplier.
2. Recover islets from the shipment on the day of islet arrival. Perform this step using a hood to minimize the chance of contamination.
   1. Cool the complete islet media (CMRL-1066, 10% (v/v) FBS, 1x Pen-Strep, 2 mM glutamine) in a refrigerator.
   2. Transfer the islets from the bottle to a 50 mL conical tube.
   3. Add 10 mL pre-cooled complete islet media to the emptied bottle to wash out the remaining islets. Transfer the media to the conical tube.
   4. Centrifuge the tube at 200 x g for 2 min to recover islets. Aspirate the supernatant leaving about 1-2 mL media with the pellet.
   5. Resuspend the islets with pre-cooled complete islet media. Based on the IEQ provided by the supplier, add 12 mL media per 5000 IEQ.
   6. Pour the islets in the media on a 10 cm non-treated tissue culture dish. Incubate overnight in a tissue culture incubator at 37 °C with 5% CO₂ in atmospheric air.
3. Dissociate islets as shown below. Perform this step following overnight incubation.
   1. Pre-warm complete islet media and cell dissociation solution.
   2. Prepare 1x PBS containing 0.04% BSA at room temperature.
   3. Count and hand-pick 200-300 islets using a P200 pipette and transfer the islets to a 15 mL conical tube containing 5 mL pre-warmed complete islet media.
   4. Collect the islets by centrifugation at 200 x g for 2 min. Gently aspirate the supernatant without disturbing the pellet on the bottom.
   5. Add 1.0 mL pre-warmed cell dissociation solution and disrupt the pellet by pipetting gently up and down. Incubate the islets at 37 °C for 9-11 min. Every 3 min pipette up and down slowly for 10 s to dissociate the cells into single cells.
   6. Once islet cells are well dissociated and the solution becomes cloudy, add 9 mL complete islet media and filter through a 30 µm cell strainer into a new 15 mL conical tube.
   7. Wash the tube and cell strainer with 2 mL complete islet media to collect the remaining islets and add to the same tube.
   8. Collect cells by centrifugation at 400 x g for 5 min.
   9. Gently aspirate media and resuspend the cell pellet in 5 mL 1x PBS containing 0.04% BSA (PBS-BSA).
   10. Filter through a new 30 µm cell strainer into a 15 mL conical tube and centrifuge at 400 x g for 5 min to collect cells.
   11. Aspirate the supernatant and resuspend the cell pellet in 200-300 µL 1x PBS-BSA solution.
   12. Measure the cell concentration and adjust volume to a final concentration of 400-500 cells/µL.

2. Single cell suspension quality control

1. Determine the cell concentration using a fluorescence-based automated cell counter³⁴.
   1. Mix 10 μL cells with 0.5 μL AO/DAPI. Pipette mix thoroughly. Load 10.5 μL onto the slide and run the cell count assay to determine count and viability.
   2. Dilute and/or filter the cell suspension as necessary based on the cell count.

3. Single cell partitioning using a microfluidic chip. Follow protocol from microfluidic chip manufacturer³⁵.

1. Bring 3' gel beads and reverse transcription (RT) reagents to room temperature (>30 min). Reconstitute the RT primer in TE buffer if needed.
2. Prepare RT master mix in a low bind tube as outlined in Table 1.
3. Determine the number of cells to be input for each sample. Calculate the cell suspension volume (X) necessary to deliver the desired target cell number. The calculated volume of nuclease-free water to add to each sample will be 33.8-X μL.
4. For each sample to be partitioned, add 33.8-X μL nuclease-free water into 0.2 mL PCR strip tube. Then, add 66.2 μL master mix to each strip tube. Do not add the cells to the strip tube at this point. Pipette gently to mix. Place the prepared strip tubes on ice.
5. Place a microfluidic chip into a chip case. Orient the chip case ensuring oil wells (row labeled 3) are closest to the person performing the experiment.
6. If running less than 8 samples, use 50% glycerol to fill unused channels in the following order:
   1. Add 90 µL of 50% glycerol into the wells in row 1 for all unused channels.
   2. Add 40 µL of 50% glycerol into the wells in row 2 for all unused channels.
   3. Add 270 µL of 50% glycerol into the wells in row 3 for all unused channels.
7. Snap the gel beads into a vortex. Vortex at full speed for 30 s. Tap the strip on the bench top several times to collect beads. Confirm that there are no bubbles present.
8. Add X μL of cells into the prepared strip tubes. Pipette to mix 5 times. Without discarding the pipette tips, transfer 90 μL of cell mixture to row 1 of the chip.
9. Wait for 30 s, then load 40 μL of gel beads to row 2. Pipette very slowly for this step. Dispense 270 μL of partitioning oil to the wells of row 3.
10. Hook the chip gasket onto the tabs of the chip holder. Place the assembled chip holder into the single cell partitioning device and press the run button.
11. Immediately remove the assembled chip holder upon run completion.
12. Remove the chip gasket from the holder, open the chip case at a 45° angle, and remove 100 μL of the emulsion from the chip into a blue plastic 96-well plate.

4. Single cell cDNA amplification. Follow protocol from microfluidic chip manufacturer³⁵.

1. Reverse transcription.
   NOTE: Perform this step under a clean PCR-only hood to prevent microbial and other contamination of the unamplified cDNA.
   1. Seal the 96-well blue plate with a foil seal on a heated plate sealer.
   2. Run the reverse transcription reaction in a thermal cycler as follows: 53 °C for 45 min à 85 °C for 5 min à 4 °C hold.
      NOTE: This is a safe stopping point. Samples can hold at 4 °C for up to 72 h.
2. Post-RT purification
   1. Bring the nucleic acid binding magnetic beads and nucleic acid size selection magnetic beads to room temperature and vortex to re-suspend. At this point, thaw the sample cleanup buffer for 10 min at 65 °C. Bring all other reagents to room temperature and vortex.
   2. Prepare the buffers as shown in Tables 2 and Tables 3.
3. Chemically break the emulsion and purify.
   1. To do so, gently remove the foil seal from the plate.
   2. Dispense 125 μL of pink emulsion-breaking reagent into each emulsion. Wait for 1 min, then transfer the entire volume to a clean 0.2 mL strip tube. Ensure that there is a layer of clear and a layer of pink in the strip tube.
   3. Remove 125 μL of the pink layer from the bottom of the strip tube without disturbing the clear layer. It is normal for a small volume (~15 μL) of the pink layer to remain in the tube.
   4. Add 200 μL of cleanup mix from Table 2 to the strip tube and incubate at room temperature for 10 min.
   5. Transfer the strip tube to a magnetic stand and allow the solution to clear. Remove the supernatant and discard, then wash the beads with 80% ethanol twice. Allow the beads to dry for 1 min.
   6. Remove the strip tube from the magnet and add 35.5 μL of elution solution from Table 3 to the beads. Pipette to resuspend the beads in the solution. Incubate for 2 min at room temperature.
   7. Transfer the strip tube to a magnetic stand and allow the solution to clear. Remove the purified cDNA from the strip tube and dispense it to clean 0.2 mL strip tubes.
4. Amplify the cDNA.
   1. Prepare amplification master mix in Table 4, below.
   2. Add 65 μL of cDNA Amplification Master Mix to each sample. Place the strip tube in a thermal cycler and run the following program: 98 °C 3 min à 15 cycles of [98 °C 15 s à 67 °C 20 s à 72 °C 1 min] à 72 °C 5 min à 4 °C hold
      NOTE: This is a safe stopping point. Samples can hold at 4°C for up to 72 h.
   3. Purify the amplified cDNA with 0.6x nucleic acid size selection magnetic beads. Wash twice with 80% ethanol and elute with 40.5 μL.
   4. Run the quality control cDNA using automated gel electrophoresis and fluorescence-based DNA quantitation assay^36,37.
      NOTE: This is a safe stopping point. Samples can hold at 4 °C for up to 72 h or at -20°C indefinitely.

5. Sequencing library construction

1. Tagmentation and clean-up of cDNA³⁸.
   1. Normalize cDNA to 50 ng in 20 μL of the total volume. Exact quantitation is critical in this step.
   2. Make the tagmentation mix in Table 5 and aliquot 30 μL to each 20 μL cDNA sample on ice. Put the samples in the thermal cycler and run the tagmentation protocol: 55 °C 5 min à 10 °C hold.
   3. Perform the clean-up of tagmented cDNA using columns³⁸. Add 180 μL DNA binding buffer to each sample. Transfer 230 μL to a spin column.
   4. Centrifuge at 1300 x g for 2 min and discard the flow-through.
   5. Wash twice with 300 μL DNA wash buffer. Centrifuge an additional 2 min at 1300 x g to ensure ethanol removal.
   6. Elute purified tagmented cDNA by adding 31 μL of elution buffer to the column and incubate at room temperature for 2 min.
   7. Centrifuge for 2 min at 1300 x g to recover the purified product.
2. Sample index PCR.
   1. Choose barcodes that do not overlap during a multiplexed sequencing run.
   2. Make a Sample Index PCR master mix as shown in Table 6.
   3. Add 60 μL of Sample Index PCR master mix to 30 μL of the purified sample.
   4. Add 10 μL of a 20 μM, 4-oligo sample index to each sample (record index used). The total reaction volume is now 100 μL.
   5. Place in a thermal cycler with the lid set to 105 °C. Run the following program: 98 °C 45 s à 12-14 cycles of [98 °C 20 s à 54 °C 30 s à 72 °C 20 s] à 72 °C 1 min à 4 °C hold.
      NOTE: This is a safe stopping point. Samples can hold at 4 °C for up to 72 h.
3. Purify libraries with double bead clean up.
   1. Add 100 μL of nucleic acid size selection magnetic beads to the sample and mix thoroughly with a pipette. Incubate at room temperature for 5 min.
   2. Transfer to a magnet and let it stand until the solution clears. Remove and discard the supernatant.
   3. Wash twice with 200 μL of 80% ethanol.
   4. Dry the beads on the magnet for 2 min. Remove from the magnet and add 50.5 μL of EB Buffer to the bead pellet. Pipette to re-suspend the beads in the buffer.
   5. Incubate at room temperature for 2 min. Transfer to a magnet and let stand for 2 min. Transfer 50 μL of eluted sample to a clean strip tube.
   6. Add 40 μL nucleic acid size selection magnetic beads to the sample and incubate at room temperature for 5 min. Transfer to a magnet and let the solution clear. Remove and discard supernatant.
   7. Wash twice with 125 μL of 80% ethanol.
   8. Dry the beads on the magnet for 2 min. Remove from the magnet and add 60.5 μL of EB Buffer to the bead pellet. Pipette to re-suspend the beads in the buffer.
   9. Incubate at room temperature for 2 min. Transfer to a magnet and let it stand for 2 min. Transfer 50 μL of eluted sample to a clean strip tube. This is the final library.
   10. Hold samples at 4 °C for up to 72 h or at -20 °C indefinitely. Note that this is a safe stopping point.
4. Quantify and run quality control of final libraries using automated gel electrophoresis and fluorescence-based DNA quantitation assay^36,37. Dilute the samples 1:10 prior to running quality control.

6. Library sequencing

1. Normalize each sample to be sequenced to 2 ng/μL and pool 3 μL of each normalized sample together.
2. Measure the pool concentration with fluorescence-based DNA quantitation assay³⁷.
3. Dilute the pool to 0.25 ng/μL.
4. Denature the pool as follows: 12 μL of diluted pooled sample (0.25 ng/μL) + 1 μL DNA control (1 nM), 2 μL EB Buffer + 5 μL NaOH (0.4N). Let this incubate for 5 min, then add 10 μL of 200 mM Tris pH 8.0.
5. Load 4.05 μL into 1345.95 μL HT1. Load 1.3 mL into the sequencer’s cartridge and run according to manufacturer’s guidelines³⁹ using a sequencing recipe with 26 cycles (Read 1) + 8 cycles (i7 Index) + 0 cycles (i5 Index) + 55 cycles (Read 2).

7. Read alignment (Supplemental File 1)

1. Run Cell Ranger (v2.0.0) to demultiplex raw base call (BCL) files generated by sequencing into FASTQ files. Align FASTQ files to human B37.3 Genome assembly and UCSC gene model to obtain expression quantification.
2. Alignment quality control.
   1. Generate alignment metrics and check Q30 bases, valid barcode fraction, cell-associated read fraction, mapped read fraction and reads detected in each cell.
   2. Examine the barcode rank plot to make sure the separation of the cell-associated barcodes and the background.

8. Data analysis (Supplemental File 2)

1. Cell quality control and preprocess.
   1. Exclude cells with < 500 detected genes, < 3000 total number of unique molecular identifier (UMI), > 0.2 viability score as previously described³². Adjust the cutoffs according to tissue and cell types.
   2. Remove doublets.
      1. Assess the five endocrine hormone genes (glucagon - GCG, insulin - INS, somatostatin - SST, pancreatic polypeptide - PPY, and ghrelin - GHRL) for bimodal expression pattern (high- and low-expression mode) using R package mclust⁴⁰.
      2. Remove cells that express more than one hormone gene, i.e., with two or more hormone genes in the high-expression mode.
   3. Normalize gene expression by the total UMI and multiply by the scale factor of 10,000 at cell level using R package Seurat⁴¹.
   4. Remove genes detected in less than 3 cells.
   5. Detect variable genes using average expression and dispersion of all cells. Adjust the cutoffs according to the tissue and cell types.
2. Perform the principal component analysis with the variable genes. Cluster cells with the selected number of principal components. Derive cell-cluster enriched genes by comparing one cell cluster with the rest of the cells.

Wyniki

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 ...

Dyskusje

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...

Materiały

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