Robust Acquisition of High Resolution Spatial Transcriptomes from Preserved Tissues with Immunofluorescence Based Laser Capture Microdissection

Abstract

The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue. However, obtaining comprehensive transcriptomic data based on established phenotypes while retaining this spatial information has been challenging. Here we present a general and robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to enable acquisition of finely resolved spatial transcriptomes with as few as tens of cells from snap-frozen or RNAlater-treated tissues, overcoming the long-standing problem of significant RNA degradation during this lengthy process. The efficacy of this approach is exemplified by the characterization of differences at the transcript isoform level between cells at the tip versus the main capillary body of the mouse small intestine lacteal. With the extensive repertoire of phenotype-specific antibodies that are presently available, our method provides a powerful means by which spatially resolved cellular states can be delineated in situ with preserved tissues. Moreover, such high quality spatial transcriptomes defined by immuno-markers can be used to compare with clusters obtained from single-cell RNAseq studies of dissociated cells as well as applied to bead-based spatial transcriptomics approaches that require such information a priori for cell identification.

Recent advances in spatially resolved transcriptomics: challenges and opportunities

“…In the study, Romanens and colleagues spatially characterized tumor and immune cells in triple-negative breast cancer. Using the immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) technique, it became easier to dissect tissues and study particular genes of cells that have functions that are highly dependent on the spatial organization in the tissue ( 21 ). In their study, the quality of RNA was immensely improved by a modified high-salt protocol and RNase inhibitor, which enabled the investigation of full-length transcripts and isoforms.…”

Time‐resolved transcriptomics of mouse gastric pit cells during postnatal development reveals features distinct from whole stomach development

“…Our lab has optimized the SCRB-seq method [19], and successfully combined it with LCM to analyse small amount of tissue cells. LCM enables the collection of precisely defined cells from frozen-section tissues with subcellular resolution in either histopathological stained or immuno-fluorescent labelled samples [21][22][23][24]. In brief, in these experiments, we obtained frozen sections of the mouse stomach at six time points following birth (namely, postnatal week 1, week 2, week 3, week 4, week 5 and week 8).…”