Lung Tissue Dissociation and Viable Single Cell Preparation for Single-Cell RNA-Seq – First Steps towards a Murine Pneumonia Atlas

Abstract

In pneumonia, lungs harbor a variety of resident and recruited cells participating in immune responses as well as maintaining organ functionality and physiology under stress. Quality and completeness of a potential murine pneumonia atlas depend on the quality and diversity of the single cell suspension input later subjected to single cell RNA sequencing (scRNAseq). We aimed to isolate as many different cell types with the highest viability and lowest stress response possible. Herewith, we adapted available protocols initially established to isolate fragile alveolar epithelial cells, to allow simultaneous isolation of endothelial cells and leukocytes. Dissociation of lung cells from their matrix into a single cell suspension was achieved by enzymatic digestion and mechanical processing, followed by dead cell removal via magnetic cell sorting. The obtained single cell suspensions were analyzed by flow cytometry for presence of doublets and dead cells in the following cellular populations: Alveolar macrophages, neutrophils, inflammatory macrophages/monocytes, eosinophils, vascular and lymphatic endothelial cells, type 1 and type 2 alveolar epithelial cells, and bronchial epithelial cells. The viability of all cellular populations analyzed were above the mandatory 80% required for single cell partitioning and barcoding, while doublet rates were in the expected range for the individual cell populations. Single cell suspension quality allowed for Gel beads in EMulsion (GEM) generation and barcoding, followed by cDNA amplification and library generation. Final conclusions with regards to the matrix dissociation steps – necessary to isolate single endothelial and epithelial cells – inducing stress responses that ultimately overwrite the cellsʼ pneumonia signature will be drawn following attentive analyses of sequences libraries.