GSE192551
Quantitative single-cell RNA-sequencing for alcohol- and formalin-fixed tissue sections with DRaqL; an efficient RNA recovery and cDNA amplification method for laser capture microdissection
Spatially resolved, single-cell transcriptome analysis of high quality remains challenging, despite the rise of high-resolution spatial transcriptomics.  Laser-capture microdissection (LCM) is widely used to isolate cells of interest from tissue sections.  Here, we developed DRaqL (Direct RNA recovery and quenching for LCM), a technique that allows efficient lysis of single, LCM-isolated cells from alcohol- and formalin-fixed tissue and stained frozen sections, and that is amenable to enzymatic reactions for cDNA amplification within the same sampling tubes.  Quantitative evaluations showed that single-cell RNA sequencing combined with DRaqL allowed transcriptomic profiling from frozen sections at an efficiency comparable with that from freshly dissociated cells, with small biases and errors in lowly expressed genes, in addition to allowing exon-exon junction profiling.  By applying this method to mouse ovarian frozen sections, we revealed a transcriptomic continuum of growing oocytes quantitatively associated with the size of oocytes and follicles, identified genes highly correlated with oocyte diameters, and detected oocyte-specific splice isoforms.  We further identified genes that were differentially expressed in granulosa cells depending on their distance from oocytes, suggesting distinct epigenetic regulatory mechanisms and cellular proliferation.  Thus, DRaqL provides an efficient cell lysis for single-cell cDNA amplification from frozen sections that is amenable to high-quality RNA sequencing analysis. Overall design: Single-cell RNA-seq analysis of fresh and LCM mouse embryonic stem cells cultured in 2i conditioned medium by DRaqL technology, and RNA-seq analysis of LCM single oocyte and granulosa cell.