Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics

J Vis Exp. 2024 Nov 8:(213). doi: 10.3791/66849.

Abstract

One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, such as combined single-cell RNA sequencing and metabolomics. The method in the current study utilizes flash-frozen pancreatic islets from fetal non-human primates collected over a span of two years for input into single-nucleus RNA sequencing and ATAC sequencing assays. The cytosolic fraction generated during nuclear extraction was retained for downstream capillary electrophoresis-mass spectrometry and subsequent metabolite quantification. This method allows for bulk analysis of metabolites that contribute to the changing transcriptomic and epigenomic landscapes within experimental conditions. It is applicable to many tissue types and maximizes the amount of information that can be extracted from samples that are not readily available. As the contribution of metabolism to the establishment of cellular identity via epigenetic modifications becomes more appreciated, techniques that allow for identifying the contribution of metabolites in specific cell types are timely and necessary.

Publication types

  • Video-Audio Media

MeSH terms

  • Animals
  • Chromatin Immunoprecipitation Sequencing / methods
  • Fetus / cytology
  • Fetus / metabolism
  • Islets of Langerhans* / cytology
  • Islets of Langerhans* / metabolism
  • Macaca mulatta
  • Metabolomics* / methods
  • Sequence Analysis, RNA / methods
  • Single-Cell Analysis / methods