Single-Nucleus RNA Sequencing from Fresh-Frozen Human Heart Tissue: Challenges and Optimization of Nuclei Isolation for the Chromium Fixed RNA Profiling (10x Genomics) v1
Year: 2026
Type: preprint
Abstract: Single-nucleus RNA sequencing (snRNA-seq) has emerged as a critical approach for transcriptomic profiling of the human heart. Frozen heart tissue presents additional challenges: the freeze-thaw cycle disrupts cytoplasmic membranes, increases cellular debris, promotes nuclear clumping through the release of genomic DNA, and compromises nuclear integrity if isolation conditions are not tightly controlled. Cardiomyocytes in particular are large, polynucleated cells with abundant cytoplasmic content that must be efficiently cleared during lysis and washing steps to yield a clean nuclei suspension. This protocol describes an optimized workflow for nuclei isolation from fresh-frozen human heart tissue, addressing these specific challenges through sequential filtration, DNase I treatment to dissolve DNA-mediated aggregates, and sucrose cushion purification to separate intact nuclei from cytoplasmic debris. Isolated nuclei are subsequently fixed and processed using the Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples (User Guide CG000527, 10x Genomics), enabling cost-effective, high-quality transcriptomic profiling of the human heart across multiple samples in a single GEM reaction.
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: National Institutes of Health
OpenAlex ID: W7161938088