High-resolution spatial RNA profiling of tissues with immuno-labeling based LCM

ABSTRACT: We report a robust protocol to effectively maintain high RNA quality for immuno-fluorescence-based laser capture microdissection (LCM), enabling finely resolved spatial transcriptome acquisition with only tens of cells from snap-frozen or RNAlater-treated tissues. With the repertoire of readily-available antibodies, this method fulfils the potential of LCM to determine native transcriptomic landscapes, as exemplified here with the characterization of mouse lacteal tip and tube cells. Methods: mRNA profiles of mouse small intestine epithelial cells (labeled by panCK) and lateal tip and tube cells (labele by Lyve1), as well as human jejunum lymphatic vessel cells were generated by deep sequencing, lacteal tip and tube samples in triplicate, using Illumina hiseq2000. The sequence reads that passed quality filters were analyzed at both the gene expression level and transcript isoform level with Stringtie. Results: Using an optimized data analysis workflow, we mapped about 15 million sequence reads per epithelial sample and about 50 million reads per lymphatic vessel cells (in cluding lacteal) to the mouse genome (GRCm38,mm10) and identified average more than 12,000 genes for each sample. RNA-seq data had a average pearson correlation higher than 0.88 for same type samples. Approximately 1.3% of the expressed genes showed differential expression between the lacteal tip and tube samples, with a log2(fold change) ≥2 or ≤-2 and padj value <0.01. This also indicate the overall sameness of lacteal tip and tube cells (the small difference is that tip cells exist filopodia structrue). Differentially expressed genes uncovered several intersting genes that reflected the potential mechanism responsible for the filopodia stucture difference. Conclusions: We have developed an easily adaptable approach that enables high-quality RNA analysis from a few 10s of cells using IF/LCM from tissues either snap-frozen or preserved with RNAlater. Such methods are uniquely required for the comprehensive delineation of spatial transcriptomic landscapes of complex tissues under near-native conditions. Only with this information can we start to unravel the basic principles that underlie the physiological and pathophysiological processes within these tissues as well as the identity and fundamental characteristics of the resident cells contained therein.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE158396 | GEO | 2022/02/25

REPOSITORIES: GEO