Comparison of optimized methodologies for isolating nuclei from esophageal tissue

Area of Science:

• Molecular Biology
• Genomics
• Tissue Analysis

Background:

• Single-nuclei RNA sequencing (snRNA-seq) enables transcriptomic analysis of frozen tissues, overcoming limitations of enzymatic dissociation.
• Esophageal tissue presents challenges due to high endogenous RNase activity.

Purpose of the Study:

• To present and optimize two methods for isolating and sequencing nuclei from esophageal tissue.
• To evaluate the efficiency and quality of nuclei isolation using commercial EZ and citric acid (CA)-based methods.

Main Methods:

• Optimization of nuclei isolation protocols for frozen esophageal tissue.
• Implementation of single-nuclei RNA sequencing using commercial EZ and CA-based kits.
• Bioinformatic analysis of sequencing data to assess library complexity and gene expression.

Main Results:

• Both EZ and CA methods yielded libraries of expected fragment lengths (EZ: 745 bp; CA: 1232 bp).
• Comparable library complexity was observed between methods (EZ: 496 transcripts/254 genes; CA: 483 transcripts/256 genes).
• The CA method showed a higher proportion of ribosomal gene reads, possibly due to co-isolated ribosomes.

Conclusions:

• Optimized snRNA-seq protocols are effective for analyzing frozen esophageal tissue, even with high RNase activity.
• The choice between EZ and CA methods depends on specific experimental requirements, as they show differences in cell type recovery.
• snRNA-seq provides valuable insights into esophageal cell lineages and tissue heterogeneity.