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Updated: Jun 21, 2026
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A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing
Published on: July 28, 2023
Protocol to maximize single-nuclei multiome library complexity for limited primary tissues via optimized sample
Faizan Uddin1, Awadhesh Pandit1, Akshay Kudpaje2
1National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru, Karnataka 560065, India.
STAR Protocols
|June 19, 2026
Summary
This study introduces a new protocol for analyzing head and neck squamous cell carcinoma (HNSCC) tissues. The method improves single-nuclei multiomic profiling by reducing debris and background noise for better results.
Area of Science:
- Oncology
- Genomics
- Molecular Biology
Background:
- Single-nuclei multiomic profiling of clinical biopsies is challenging due to limited tumor mass and high debris.
- Head and neck squamous cell carcinoma (HNSCC) presents unique dissociation and purification difficulties.
Purpose of the Study:
- To develop an optimized protocol for dissociating HNSCC and matched-normal tissues for single-nuclei multiomic analysis.
- To enhance the quality and complexity of single-nuclei assay for transposase-accessible chromatin sequencing (sn-ATAC) and gene expression (GEX) libraries.
Main Methods:
- A mechanical-enzymatic dissociation workflow was employed for HNSCC and matched-normal tissues.
- Red blood cell (RBC) lysis and DNase-I treatment were used to minimize ambient nucleic acid contamination.
- Density gradient centrifugation was utilized for efficient removal of cellular debris.
- Intact nuclei were isolated for modified sn-ATAC and 3'-GEX library preparation, including indexing PCR and size selection.
Main Results:
- The protocol effectively dissociates HNSCC and normal tissues while preserving nuclear integrity.
- Ambient nucleic acid background was significantly reduced through RBC lysis and DNase-I treatment.
- Cellular debris was efficiently removed via density gradient centrifugation, improving sample purity.
- Optimized sn-ATAC and 3'-GEX library preparation maximized library complexity.
Conclusions:
- This protocol provides a robust method for single-nuclei multiomic profiling of HNSCC biopsies.
- The described techniques enhance data quality by reducing noise and increasing library complexity.
- This workflow facilitates deeper investigation into the molecular landscape of HNSCC.
