Single-cell transcriptomics from human pancreatic islets: sample preparation matters

• Published in: Biology methods and protocols 2020, Vol.5 (1), p.bpz019-bpz019
• Main Authors: Bonnycastle, Lori L, Gildea, Derek E, Yan, Tingfen, Narisu, Narisu, Swift, Amy J, Wolfsberg, Tyra G, Erdos, Michael R, Collins, Francis S
• Format: Article
• Language: English
• Subjects: Methods Manuscript
• ISSN: 2396-8923; 2396-8923
• DOI: 10.1093/biomethods/bpz019

Abstract Single-cell RNA sequencing (scRNA-seq) of human primary tissues is a rapidly emerging tool for investigating human health and disease at the molecular level. However, optimal processing of solid tissues presents a number of technical and logistical challenges, especially for tissues that are only available at autopsy, which includes pancreatic islets, a tissue that is highly relevant to diabetes. To assess the possible effects of different sample preparation protocols on fresh islet samples, we performed a detailed comparison of scRNA-seq data generated with islets isolated from a human donor but processed according to four treatment strategies, including fixation and cryopreservation. We found significant and reproducible differences in the proportion of cell types identified, and more minor effects on cell-specific patterns of gene expression. Fresh islets from a second donor confirmed gene expression signatures of alpha and beta subclusters. These findings may well apply to other tissues, emphasizing the need for careful consideration when choosing processing methods, comparing results between different studies, and/or interpreting data in the context of multiple cell types from preserved tissue.