Improving fibroblast characterization using single-cell RNA sequencing: an optimized tissue disaggregation and data processing pipeline.

Single-cell RNA sequencing (scRNA-Seq) provides a valuable platform for characterising multicellular ecosystems. Fibroblasts are a heterogeneous cell type involved in many physiological and pathological processes, but remain poorly-characterised. Analysis of fibroblasts is challenging: these cells are difficult to isolate from tissues, and are therefore commonly under-represented in scRNA-seq datasets. Here, we describe an optimised approach for fibroblast isolation from human lung tissues. We demonstrate the potential for this procedure in characterising stromal cell phenotypes using scRNA-Seq, analyse the effect of tissue disaggregation on gene expression, and optimise data processing to improve clustering quality. Overall design: mRNA profiles of human lung samples (tumour, inflamed and normal lung) from 3 patients.