Protocol for high-quality single-cell RNA-seq from tissue sections with DRaqL.

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Single-cell RNA sequencing (scRNA-seq) is crucial for understanding cellular heterogeneity.
• Analyzing transcriptomics directly from tissue sections presents technical challenges.
• Laser capture microdissection (LCM) facilitates isolation of specific cells from tissues.

Purpose of the Study:

• To present a detailed protocol for scRNA-seq from fixed murine ovarian tissue sections.
• To enable high-quality transcriptomic profiling of single cells isolated by LCM.
• To offer adaptable methods for both alcohol-fixed and formalin-fixed tissues.

Main Methods:

• The protocol integrates direct RNA recovery and quenching for LCM (DRaqL) with Smart-seq2.
• Key steps include cell lysis, cDNA amplification, and sequencing library preparation.
• An optional procedure using protease is provided for formalin-fixed samples.

Main Results:

• The DRaqL-Smart-seq2 protocol yields high-quality RNA sequencing data from single cells.
• The method is effective for analyzing transcriptomes of cells from alcohol-fixed murine ovarian sections.
• An adapted protocol (DRaqL-Protease-Smart-seq2) is suitable for formalin-fixed tissues.

Conclusions:

• The presented DRaqL-Smart-seq2 protocol offers a robust method for single-cell transcriptomics from LCM-isolated cells in fixed tissues.
• This technique enhances the ability to study cellular composition and gene expression within complex tissue architectures.
• The protocol provides a valuable tool for researchers in reproductive biology and beyond.