Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics
November 8th, 2024
This protocol describes procedures to isolate high-quality nuclei from frozen non-human primate pancreatic islets for use in single-nucleus simultaneous RNA sequencing and ATAC sequencing while preserving the bulk cytosolic fraction for metabolomic analyses from the same samples.
We utilize a highly translational non-human primate model to assess how maternal diet and medication consumption during pregnancy influence the embryonic development and postnatal function of pancreatic islets in the offspring. Developmentally programmed changes could alter disease susceptibility later in life. A challenge when working with hard-to-acquire samples such as those from non-human primate is evaluating multiple questions from the same individual efficiently.
Unfortunately, simultaneously extracting information from the metabolome and genome can be difficult due to the incompatibility of assay reagent and time-sensitive degradation of molecules to be quantified. Our protocol allows for extrapolation of metabolite abundance, chromatin accessibility, and differential gene expression from the same starting material. This multi-layered approach provides insight into how metabolism may alter the genome in pancreatic islets.
To begin, thaw the isolated Rhesus-Macaque pancreatic islet tissue on ice, add 500 microliters of 0.1x lysis buffer to the tissue pellet. Using a disposable RNA's free pellet pestle, gently homogenize the tissue 15 times on ice. Incubate the sample on ice for 10 minutes.
Then add 500 microliters of wash buffer to the homogenate and gently mix with pipette on ice. Prime a 30-micrometer pre-separation filter with 300 milliliters of wash buffer and filter one milliliter of cellular homogenate into a five-milliliter tube. Spin the filtered homogenate at 500 G for five minutes in a swinging bucket centrifuge at 4 degrees Celsius.
Using a pipette, transfer one milliliter of the supernatant to a cryovial on ice and immediately freeze it at 80 degrees Celsius for future metabolomics experiments. Add one milliliter of wash buffer dropwise to the nuclei pellet to resuspend it. Spin the washed pellet at 1000 G for five minutes in a swinging bucket centrifuge at 4 degrees Celsius.
After repeating the wash, resuspend the nuclei in one milliliter of wash buffer. With a pipette, mix 10 microliters each of nuclei suspension and 0.4%trypan blue in a separate tube, and add the mixture to an automated cell counter slide. Finally, using a cell counter, determine the nuclei concentration.
An ideal intact nucleus was obtained for the sequencing experiments. Based on gene expression studies, the UMAP clustering of cellular subtypes within isolated fetal non-human primate islets was obtained. Islet metabolite abundance such as dihydroxyacetone phosphate, glycerol 3-phosphate, 2-oxoglutaric, creatine, and glutathione was obtained using the cytosolic fractions.
Overview
This protocol outlines methods to isolate high-quality nuclei from frozen non-human primate pancreatic islets for simultaneous RNA sequencing and ATAC sequencing. It also preserves the bulk cytosolic fraction for metabolomic analyses, enabling a comprehensive understanding of metabolic and genomic interactions.
Key Study Components
Area of Science
- Neuroscience
- Biology
- Metabolomics
Background
- Non-human primate models are crucial for studying human disease mechanisms.
- Maternal diet and medication can influence offspring pancreatic islet development.
- Simultaneous analysis of metabolome and genome is challenging due to sample degradation.
- This study addresses the need for efficient multi-layered analyses from limited samples.
Purpose of Study
- To assess the impact of maternal factors on embryonic pancreatic islet development.
- To explore how metabolic changes can influence genomic expression.
- To develop a protocol that allows for comprehensive analysis from the same sample.
Methods Used
- Isolation of nuclei from pancreatic islets.
- Simultaneous RNA sequencing and ATAC sequencing.
- Preservation of cytosolic fractions for metabolomic studies.
- Multi-layered analysis to assess metabolite abundance and gene expression.
Main Results
- Successful isolation of high-quality nuclei and cytosolic fractions.
- Insights into how maternal diet affects pancreatic islet function.
- Demonstrated feasibility of simultaneous genomic and metabolomic analyses.
- Revealed potential links between metabolism and gene expression in islets.
Conclusions
- This protocol enables comprehensive analysis of pancreatic islets.
- Findings may inform future studies on disease susceptibility.
- Highlights the importance of maternal influences on offspring health.
Frequently Asked Questions
Executive Industry Relevance
Integrating single-nucleus RNA-seq, ATAC-seq, and bulk metabolomics from frozen non-human primate islets addresses a critical bottleneck in multi-omic discovery using rare or precious samples. This approach enables comprehensive molecular profiling from limited material, supporting predictive confidence in early target validation and mechanistic de-risking. The protocol maximizes data yield per sample, informing risk-adjusted portfolio decisions in metabolic and developmental disease research.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables interrogation of metabolic and epigenetic mechanisms underlying cellular identity in disease-relevant systems.
- Supports functional target validation by linking metabolite abundance to gene expression and chromatin accessibility.
- Facilitates mechanistic de-risking by integrating multi-layered molecular data from the same biological source.
Screening & Assay Development
- Prepares validated nuclei and cytosolic fractions for downstream high-throughput sequencing and metabolomics workflows.
- Improves assay reproducibility and standardization by minimizing batch effects across multi-cohort studies.
- Enables quantitative, multi-omic outputs for robust compound or perturbation evaluation.
Translational & Preclinical Research
- Aligns molecular readouts with disease-relevant metabolic and epigenetic biomarkers in translational models.
- Supports continuity from discovery through preclinical validation by leveraging non-human primate systems.
- Provides predictive insights into developmental programming and disease susceptibility mechanisms.
Pipeline & Workflow Integration
This protocol bridges early discovery, target validation, and translational research by enabling multi-omic profiling from rare tissue samples.
- Discovery Biology: Supports hypothesis testing on metabolic-epigenetic interactions in islet development.
- Screening: Delivers reproducible nuclei and metabolite preparations for scalable omics assays.
- Analytics: Provides quantitative gene expression, chromatin accessibility, and metabolite abundance data for comparative analyses.
- Translational Research: Connects molecular findings to disease-relevant phenotypes in non-human primate models.
- Enterprise Reuse: Establishes a reusable workflow for maximizing data extraction from limited or archival samples.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in early-stage research.
- Operational Value: Standardizes multi-omic sample preparation and minimizes batch effects.
- Strategic Value: Enables more informed go/no-go decisions and capital-efficient use of rare samples.
- Portfolio Impact: Supports risk-adjusted prioritization of metabolic and developmental disease programs.
Implementation Considerations
- Requires expertise in nuclei isolation, single-nucleus sequencing, and metabolomics workflows.
- Demands access to high-throughput sequencing and mass spectrometry infrastructure.
- Necessitates rigorous cross-team standardization to control for batch effects.
- Adaptable to other tissue types but may require protocol optimization for different sample matrices.
- Sample scarcity and degradation sensitivity impose practical limitations on throughput and timing.
