Assessing the Influence of Selected Permeabilization Methods on Lymphocyte Single-Cell Multi-Omics

Area of Science:

• Immunology
• Genomics
• Proteomics
• Single-cell analysis

Background:

• Single-cell multi-omics is crucial for understanding complex immune responses.
• Current methods often introduce bias by combining omics data from different sample parts.
• Integrating intracellular proteomics with other omics remains a significant challenge.

Purpose of the Study:

• To assess the impact of minimal fixation and permeabilization on transcriptomics and proteomics data quality.
• To evaluate these methods using the BD Rhapsody™ Single-Cell Analysis System.
• To optimize protocols for combined intracellular proteomics and other omics in immune studies.

Main Methods:

• Evaluation of established minimal fixation and permeabilization protocols.
• Generation of transcriptomics and proteomics data using the BD Rhapsody™ system.
• High-throughput sequencing (HiseqX platform) for multi-omics data analysis.
• Unsupervised clustering to analyze immune cell populations (e.g., T cells).

Main Results:

• High-throughput sequencing is essential for combining multi-omics data from single cells.
• Fixation/permeabilization minimally affected unstimulated cell transcriptomes but impacted overall detection.
• Approximately 60% of the transcriptomic signature of immune stimulation was retained.
• A modified fixation/permeabilization method reduced transcriptomic loss and improved proteomic accuracy.

Conclusions:

• The study supports the integration of intracellular proteomics with other omics data.
• Optimized fixation and permeabilization are key for accurate multi-omics profiling.
• Further development with oligonucleotide-tagged antibodies is needed for robust intracellular proteomic analysis.