Single Cell Isolation from Human Diabetic Fibrovascular Membranes for Single-Cell RNA Sequencing.

Abstract

Single-cell transcriptomic analyses have emerged as very powerful tools to query the gene expression changes at the single-cell level in physiological and pathological conditions. The quality of the analysis is heavily dependent on tissue digestion protocols, with the goal of preserving thousands of single live cells to submit to the subsequent processing steps and analysis. Multiple digestion protocols that use different enzymes to digest the tissues have been described. Harsh digestion can damage certain cell types, but this might be required to digest especially fibrotic tissue as in our experimental condition. In this paper, we summarize a collagenase type I digestion protocol for preparing the single-cell suspension from fibrovascular tissues surgically removed from patients with proliferative diabetic retinopathy (PDR) for single-cell RNA sequencing (scRNA-Seq) analyses. We also provide a detailed description of the data analysis that we implemented in a previously published study. Key features • Single-cell suspension from fibrovascular membranes isolated from PDR patients. • Single-cell RNA sequencing analyses performed using Seurat package in RStudio. • Trajectory analyses or pseudotime analyses to study the trajectory over (pseudo)time of specific cell types. • This protocol requires Illumina HiSEQ4000 instrument and knowledge of R and RStudio language for the analyses.