High-quality single-cell transcriptomics from ovarian histological sections during folliculogenesis

Area of Science:

• Molecular Biology
• Genomics
• Developmental Biology

Background:

• High-quality single-cell RNA sequencing (RNA-seq) with spatial resolution from tissue sections is technically challenging.
• Existing methods often face limitations in efficiency and applicability to various fixation methods.

Purpose of the Study:

• To develop an efficient experimental approach for high-quality single-cell RNA sequencing from tissue sections.
• To enable transcriptomic profiling from both alcohol-fixed and formalin-fixed tissues.
• To investigate the heterogeneity of the ovarian follicle transcriptome.

Main Methods:

• Development of DRaqL (direct RNA recovery and quenching for laser capture microdissection) for efficient cell lysis and direct cDNA amplification.
• Integration of DRaqL with single-cell RNA sequencing on alcohol-fixed and formalin-fixed tissue sections.
• Application of the method to mouse ovarian sections for transcriptomic analysis.

Main Results:

• DRaqL achieved transcriptomic profiling efficiency comparable to freshly dissociated cells from alcohol-fixed sections.
• Effective exon-exon junction profiling was maintained.
• Combined DRaqL with protease treatment enabled robust single-cell transcriptome analysis from formalin-fixed sections.
• Identified oocyte-specific splice isoforms and an anomaly in the size-transcriptome relationship related to oocyte growth retardation.
• Detected differentially expressed genes in granulosa cells correlated with proximity to oocytes, suggesting epigenetic and cell-cycle regulation.

Conclusions:

• DRaqL is a versatile and efficient method for high-quality single-cell RNA-seq from tissue sections.
• The approach facilitates the study of histological heterogeneity within the folliculogenic transcriptome.
• Provides new insights into germ-soma interactions and gene regulation during oocyte development.