May 21, 2026

Single-Nucleus RNA Sequencing from Fresh-Frozen Human Heart Tissue: Challenges and Optimization of Nuclei Isolation for the Chromium Fixed RNA Profiling (10x Genomics)

DOI
https://dx.doi.org/10.17504/protocols.io.n92ld4oz7l5b/v1
Single-Nucleus RNA Sequencing from Fresh-Frozen Human Heart Tissue: Challenges and Optimization of Nuclei Isolation for the Chromium Fixed RNA Profiling (10x Genomics)
  • 1Division of Pulmonary, Critical Care, & Sleep Medicine, Department of Internal Medicine, The Ohio State University, Wexner Medical Center, Columbus, OH 43210, USA
  • TriState SenNet
  • Cellular Senescence Network (SenNet) Method Development Community
Lorena Rosas
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ld4oz7l5b/v1
Protocol Citation: Natalia-Del Pilar Vanegas, Carmen E. Badell-Kestler, Tamer Sleik, Victor Peters, Lorena Rosas, Ana L. Mora, Mauricio Rojas 2026. Single-Nucleus RNA Sequencing from Fresh-Frozen Human Heart Tissue: Challenges and Optimization of Nuclei Isolation for the Chromium Fixed RNA Profiling (10x Genomics). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld4oz7l5b/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 20, 2026
Last Modified: May 21, 2026
Protocol  Integer ID: 317483
Keywords: snRNA-seq, Fresh-frozen human heart tissue, Nuclei isolation, 10x Genomics, chromium fixed rna profiling reagent kit, chromium fixed rna profiling, quality transcriptomic profiling of the human heart, workflow for nuclei isolation, frozen human heart tissue, nucleus rna, frozen heart tissue, optimization of nuclei isolation, critical approach for transcriptomic profiling, clean nuclei suspension, transcriptomic profiling, isolated nuclei, nuclei isolation, polynucleated cells with abundant cytoplasmic content, cardiomyocyte, intact nuclei from cytoplasmic debris, polynucleated cell, intact nuclei, 10x genomic, abundant cytoplasmic content, nuclei, genomic dna, nuclear integrity if isolation condition, dnase, nuclear clumping through the release
Funders Acknowledgements:
TriState SenNET (Lung and Heart) Tissue Map and Atlas consortium - NIH
Grant ID: U54AG075931
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.
Guidelines
Tissue collection may only proceed with prior approval from the user’s Institutional Ethics Board (IEB) or an equivalent ethics committee.
Materials
Nuclei isolation reagents:
- Tris-HCl, pH 7.4 (1 M)
- NaCl (5 M)
- MgCl₂ (1 M)
- IGEPAL CA-630 (10%)
- Digitonin (5%)
- BSA, Nuclease-Free (10%)
- DTT (1000 mM; store at −20 °C)
- RNase Inhibitor (40 U/µL)
- Tween-20 (10%)
- DNase I, RNase-free (1 U/µL)
- EDTA (50 mM)
- Sucrose (MW 342.3 g/mol)
- Nuclease-free water
- 1× Phosphate-Buffered Saline, without Calcium and Magnesium
Fixation and library preparation (10x Genomics):
- Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (PN-1000414)
- Chromium Fixed RNA Profiling Reagent Kit for Multiplexed Samples — 16 Probe Barcodes (PN-1000420 or PN-1000456)
- Chromium Next GEM Chip Q (PN-2000338)
- Dual Index Kit TT Set A (PN-3000431)
- Chromium X/iX (PN 1000326)
Equipment and consumables:
- Cordless pellet pestle motor and microcentrifuge tubes
- 30 µm cell strainer
- 20 µm cell strainer
- Wide-bore 1000 µL and 200 µL pipette tips
- Temperature-controlled microcentrifuge (4 °C capable)
- Thermocycler supporting uniform heating of 100 µL volumes
- Countess III FL Automated Cell Counter or equivalent
- Propidium Iodide (for fixed nuclei viability staining)
A. Buffer Preparation. Prepare fresh on day of use and keep at 4 °C
Lysis Buffer. Use 1 mL per 50 mg tissue
Reagent Concentration Volume
Tris-HCl (pH 7.4) 1 M 20 µL
NaCl 5 M 4 µL
MgCl₂ 1 M 6 µL
IGEPAL 10% 10 µL
Digitonin (pre-dissolved at 65 °C) 5% 4 µL
BSA 10% 200 µL
DTT 1000 mM 2 µL
Nuclease-free water 1,754 µL
Total 2,000 µL
Wash / Resuspension Buffer
Reagent Concentration Volume
BSA 10% 500 µL
RNase Inhibitor 40 U/µL 25 µL
DTT 1000 mM 5 µL
Tween-20 10% 50 µL
Tris-HCl 1 M 50 µL
NaCl 5 M 10 µL
MgCl₂ 1 M 15 µL
Nuclease-free water 4,345 µL
Total 5,000 µL
0.25 M Sucrose Buffer — dissolve 4.28 g sucrose in 50 mL Wash Buffer; keep cold.
B. Nuclei Isolation
Keep all reagents and samples at 4 °C. Use wide-bore tips at every pipetting step.
  1. Start with 50 mg of pulverized frozen tissue (cryopulverized under liquid nitrogen). Maintaining the tissue frozen until lysis is critical.
  2. Add 200 µL lysis buffer to the pulverized tissue. Homogenize with cordless pellet pestle motor for ~30 seconds. Avoid over-homogenization.
  3. Add remaining 800 µL lysis buffer (total 1 mL). Incubate on ice for exactly 1 minute. Prolonged lysis in the presence of IGEPAL and Digitonin risks nuclear lysis.
  4. Filter lysate through a 30 µm cell strainer to remove large myofibrillar debris and undigested tissue fragments, which are abundant in heart tissue.
  5. Vortex filtered lysate briefly (10 sec) to dissociate loose aggregates before centrifugation.
  6. Centrifuge at 500 × g, 3 min, 4 °C.
  7. Discard supernatant completely.
  8. Resuspend pellet in 1 mL wash buffer using gentle wide-bore pipetting (5–6 strokes).
  9. Centrifuge at 300 × g, 5 min, 4 °C.
  10. Discard supernatant.
  11. Repeat once (2 washes total). The lower g-force in wash steps favors nuclei recovery over debris pellet down. RNase Inhibitor in the wash buffer protects nuclear RNA throughout these steps.
  12. Resuspend pellet in 1 mL wash buffer.
  13. Add 10 µL DNase I (1 U/µL). Mix by inverting the tube 2–3× only — do not vortex, as this disrupts nuclei.
  14. Incubate on ice for 5 minutes. DNase I treatment is especially critical for heart tissue.
  15. Stop DNase I activity by adding 10 µL 50 mM EDTA. EDTA chelates the Mg²⁺ cofactor required for DNase I catalysis.
  16. Filter suspension through a 20 µm strainer to remove residual debris and any remaining aggregates not dissolved by DNase I treatment.
  17. Centrifuge at 300 × g, 5 min, 4 °C.
  18. Discard supernatant.
  19. Resuspend pellet in 1 mL 0.25 M sucrose buffer.
  20. Centrifuge at 500 × g, 5 min, 4 °C. The sucrose cushion provides density-based separation.
  21. Discard the top layer carefully.
  22. Retain the pellet.
  23. Resuspend nuclei in an appropriate volume of Wash Buffer.
  24. Assess nuclei by microscopy before counting: nuclei from heart tissue should appear round and uniform with minimal debris. If significant clumping persists, perform an additional DNase I treatment (5 min on ice) and re-filter through 20 µm.
  25. Count nuclei using Countess III FL with Propidium Iodide staining. Target: ≥ 500,000 intact nuclei at ≥ 70% viability for fixation input.
C. Nuclei Fixation and Permeabilization
  1. Perform fixation according to Demonstrated Protocol CG000478 (Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling).
  2. Recount fixed nuclei using Countess II FL with Ethidium Homodimer-1 before proceeding.
D. Probe Hybridization
  1. Prepare a Hybridization Mix for each sample by combining the fixed nuclei suspension with the sample-specific Probe Barcode set (LHS + RHS probe pairs; Chromium Next GEM Single Cell Fixed RNA Human Transcriptome Probe Set, PN-1000420/1000456).
  2. After hybridization, add Post-Hybridization (Post-Hyb) Buffer and wash to remove unbound probes.
  3. Recount fixed nuclei using Countess II FL with Ethidium Homodimer-1.
  4. Resuspend each hybridized sample in the volume specified for pooling.
E. Sample Pooling and Cell Counting
  1. Pool up to 16 hybridized samples in equal proportions. Consult Pooling Workbook CG000565 for equalization strategies.
  2. Centrifuge pooled sample at 300 × g, 5 min, 4 °C. Resuspend in GEM loading volume specified in CG000527.
  3. Count pooled nuclei with Countess III FL. Adjust to target input concentration for Chromium Chip Q loading.
F. GEM Generation, Ligation, and Library Construction
  1. Proceed immediately to GEM generation per CG000527 on Chromium X/iX.
  2. Construct Gene Expression libraries by PCR amplification using Dual Index Kit TT Set A. Assess library quality with Bioanalyzer (expected size ~200–300 bp).
  3. Sequence on an Illumina platform: 28 cycles (R1) + 10 cycles (i7) + 10 cycles (i5) + 90 cycles (R2). Target ≥ 15,000 reads per nucleus per sample.
G. Data Analysis
Raw sequencing data from the Chromium Fixed RNA Profiling (Flex) libraries are processed using the cellranger multi pipeline — the only supported pipeline for Flex v1 multiplexed datasets. Analysis is performed via 10x Genomics Cloud Analysis (cloud.10xgenomics.com), a web-based platform that provides access to Cell Ranger pipelines.
  1. Upload FASTQ files using the Cloud CLI tool.
  2. After upload, FASTQ sets are automatically grouped.
  3. Assign the library type as "Flex Gene Expression" for each FASTQ set in the project interface before creating an analysis.
  4. Select all FASTQ files from a single GEM well and click "Create New Analysis".
  5. Specify the following Flex-specific parameters: (i) reference genome — human GRCh38-2024-A; (ii) probe set reference matching the kit used (PN-1000420 or PN-1000456); (iii) the Probe Barcode ID assigned to each sample during hybridization, for per-sample demultiplexing.
  6. Each GEM well constitutes one analysis. Cell Ranger multi automatically demultiplexes all pooled samples into per-sample outputs using the Probe Barcode sequences.
  7. The latest version of Cell Ranger is used by default on Cloud Analysis.
Key outputs and quality control:
  1. web_summary.html — interactive per-sample QC report. Review: estimated number of nuclei, median genes per nucleus, sequencing saturation, fraction of reads confidently mapped to the probe set, and the knee plot for cell calling. Flag samples with saturation < 50% or median genes < 1,000 for re-sequencing.
  2. sample_cloupe.cloupe — Loupe Browser file for interactive visualization of UMAP projections, unsupervised clusters, and per-gene expression. Open in Loupe Browser (free desktop application, Windows/macOS) for exploratory cell type annotation without coding.
  3. filtered_feature_bc_matrix/ — per-sample feature-barcode count matrices (barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz). These are the primary input for downstream analysis in R (Seurat) or Python (Scanpy/AnnData).
Protocol references
  1. 10x Genomics. Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling. Demonstrated Protocol CG000478 Rev D. 10x Genomics, Inc.; 2024.
  2. 10x Genomics. Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples. User Guide CG000527 Rev D. 10x Genomics, Inc.; 2023.
  3. 10x Genomics. Cloud Analysis: Web-based Cell Ranger Pipeline Platform. cloud.10xgenomics.com. Accessed 2025.