Single-nucleus total RNA sequencing of formalin-fixed paraffin-embedded samples using snRandom-seq

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Formalin-fixed paraffin-embedded (FFPE) tissues are a rich source of clinical data but difficult for single-nucleus RNA sequencing.
• Existing high-throughput single-cell/single-nucleus RNA sequencing (sc/snRNA-seq) methods face challenges with FFPE sample quality and RNA integrity.

Purpose of the Study:

• To develop and validate a novel method for single-nucleus RNA sequencing (snRNA-seq) specifically optimized for FFPE tissues.
• To overcome limitations in RNA capture and detection in archived clinical samples.

Main Methods:

• Developed snRandom-seq, a droplet- and random primer-based technology for FFPE snRNA-seq.
• Protocol includes single-nucleus isolation, in situ DNA blocking, reverse transcription, dA tailing, barcoding, and library preparation.
• Utilizes random primers for comprehensive total RNA capture.

Main Results:

• snRandom-seq achieved a low doublet rate of 0.3%.
• Demonstrated increased RNA coverage and enhanced detection of non-coding and nascent RNAs compared to other methods.
• The entire protocol is completed within 4 days.

Conclusions:

• snRandom-seq is a powerful and efficient platform for snRNA-seq analysis of FFPE clinical specimens.
• This technology expands the utility of archived tissues for studying complex biological systems and diseases.
• Enables deeper insights into cellular heterogeneity and molecular mechanisms from historical patient cohorts.