Next generation sequencing, especially single cell RNA Sequencing (scRNA-Seq) using enzymatic digestion, has revolutionised our ability to study tissues. However, several problems remain as these techniques have traditionally been used for tissues from which substantial cell numbers are easily liberated. Tissues with low-cellularity and a substantial extracellular matrix present a significant challenge for enzymatic scRNA-Seq approaches due to stress responses to digestion. Here, we describe a robust protocol for the isolation of nuclei from snap frozen fibrous tissues, including tendon and ligament, for use in single nucleus RNA Sequencing. This use of snap-frozen tissue enables tissues that are collected from different centres or/and over a period of time to be processed together. Therefore, this is not only convenient for prevention of a stress response and for collecting and processing tissue (especially tissues that are not very accessible), but it might also reduce batch effects by preventing the need to process tissues fresh on different days. This protocol was adapted from Slyperet al.(2020) Nature Medicine.
Single Nuclei RNA Sequencing of Tendon Tissue - small tissue biopsies v1 (protocols.io.9sth6en)
