Single nuclei extraction has become a crucial technique in molecular biology, particularly for single-cell and spatial transcriptomics applications. While it offers unique advantages over single-cell isolation, such as enabling the analysis of frozen tissues or samples where whole-cell dissociation is challenging, it also presents significant obstacles. Among the most pressing challenges are minimizing debris and reducing the occurrence of doublets. Addressing these issues is essential for obtaining high-quality sequencing data and accurate downstream analysis.

1. Debris Formation

Debris in single nuclei suspensions arises primarily due to incomplete lysis, mechanical damage, or improper filtration. This extraneous material can interfere with downstream processes, leading to inaccurate quantification and increased background noise in sequencing data. Common sources of debris include:

• Overly harsh lysis conditions, causing fragmentation of nuclear material
• Residual cytoplasmic content that is not fully removed
• Cellular organelles and apoptotic bodies that persist in the suspension

2. Doublet Formation

Doublets, where two nuclei remain attached or are co-encapsulated, can significantly impact data interpretation by confounding true biological signals. Doublet formation often results from:

• Inadequate dissociation techniques, leading to clumped nuclei
• Excessive pipetting or mechanical stress that promotes adhesion
• Suboptimal sample preparation, where nuclei remain embedded in tissue remnants

Optimizing Lysis Conditions :Finding the balance between effective lysis and nuclear integrity is crucial. Consider the following approaches:

• Gentle lysis buffers: Using optimized detergents and salt concentrations can help break open cells while preserving nuclei.
• Titration of lysis time: Prolonged exposure to lysis buffers can degrade nuclei; therefore, empirical testing to determine the minimal effective time is essential.
• Temperature control: Keeping samples on ice throughout processing minimizes degradation and prevents excess debris.

Proper filtration and washing ensure a clean nuclei suspension free of cytoplasmic debris and unwanted cellular fragments.

• Use of low-speed centrifugation: Helps separate nuclei from smaller debris without compromising nuclear integrity.
• Sequential filtration: Passing the suspension through cell strainers (e.g., 40-µm filters) removes larger fragments while retaining intact nuclei.
• Additional washing steps: Multiple washes with buffer solutions help remove residual contaminants and organelles.

To reduce the occurrence of doublets, consider the following approaches:

• Optimize dissociation techniques: Use enzymatic digestion only when necessary and ensure gentle trituration to avoid clumping.
• FACS-based doublet exclusion: Fluorescence-activated cell sorting (FACS) can be employed to remove doublets based on nuclear size and fluorescence intensity.
• Magnetic bead separation: Using magnetic beads targeted to specific nuclear markers can aid in purifying single nuclei populations.
• Careful pipetting: Avoid over-agitating nuclei suspensions, as excessive shear forces can promote doublet formation.

Performing quality control assessments at multiple steps in the protocol can help ensure a high-quality nuclei suspension.

• Microscopy analysis: Staining with DAPI or other nuclear dyes allows visualization of debris levels and doublets.
• Flow cytometry: Can quantify nuclear integrity, size distribution, and debris levels before proceeding to sequencing.
• RNA quality assessment: Ensuring extracted nuclei yield high-quality RNA is critical for downstream applications.

Single nuclei extraction presents inherent challenges, but with optimized protocols, it is possible to minimize debris and doublets effectively. By refining lysis conditions, implementing thorough filtration and washing steps, and employing strategies to prevent and remove doublets, researchers can ensure high-quality nuclei suspensions for sequencing and other applications. Continuous improvements in extraction methodologies will enhance the reliability of single nuclei studies, paving the way for deeper insights into complex biological systems.