High-throughput RNA sequencing of paraformaldehyde-fixed single cells

Area of Science:

• Molecular Biology
• Genomics
• Virology

Background:

• Current single-cell RNA sequencing methods face limitations with paraformaldehyde fixation, hindering studies requiring intracellular staining, cell sorting, or pathogen inactivation.
• Paraformaldehyde fixation is crucial for preserving cellular structures and inactivating pathogens but is often incompatible with high-throughput single-cell RNA sequencing.

Purpose of the Study:

• To develop a high-throughput single-cell RNA sequencing method compatible with paraformaldehyde-fixed cells.
• To enable the analysis of rare cell subpopulations and pathogenic samples using single-cell transcriptomics.

Main Methods:

• FD-seq (Fixed Droplet RNA sequencing) was developed for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized, and sorted single cells.
• RNA integrity and gene expression levels were assessed after fixation and permeabilization.
• FD-seq performance was compared to methanol fixation in terms of gene and transcript detection.

Main Results:

• FD-seq preserves RNA integrity and relative gene expression levels in fixed and permeabilized cells.
• FD-seq detects a higher number of genes and transcripts compared to methanol fixation.
• Application of FD-seq identified TMEM119 as a potential host factor in KSHV reactivation and revealed upregulated pro-inflammatory pathways in OC43-infected cells.

Conclusions:

• FD-seq overcomes limitations of existing methods, allowing for droplet-based RNA sequencing of fixed cells.
• The method facilitates the integration of phenotypic and transcriptomic data in rare cell subpopulations.
• FD-seq supports the analysis of pathogenic samples while ensuring their preservation and inactivation.