Practical Considerations for Complex Tissue Dissociation for Single-Cell Transcriptomics.

Single-cell and single-nucleus RNA sequencing have revolutionized biomedical research, allowing analysis of complex tissues, identification of novel cell types, and mapping of development as well as disease states. Successful application of this technology critically relies on the dissociation of solid organs and tissues into high-quality single-cell (or nuclei) suspensions.In this chapter, we examine several key aspects of the tissue handling workflow that need to be considered when establishing an efficient tissue processing protocol for single-cell RNA sequencing (scRNA-seq). These include tissue collection, transport, and storage, as well as the choice of the dissociation conditions. We emphasize the importance of the tissue quality check and discuss the advantages (and potential limitations) of tissue cryopreservation. We provide practical tips and considerations on each of the steps of the processing workflow, and comment on how to maximize cell viability and integrity, which are critical for obtaining high-quality single-cell transcriptomic data.

UBR5 Is Coamplified with MYC in Breast Tumors and Encodes an Ubiquitin Ligase That Limits MYC-Dependent Apoptosis.

For maximal oncogenic activity, cellular MYC protein levels need to be tightly controlled so that they do not induce apoptosis. Here, we show how ubiquitin ligase UBR5 functions as a molecular rheostat to prevent excess accumulation of MYC protein. UBR5 ubiquitinates MYC and its effects on MYC protein stability are independent of FBXW7. Silencing of endogenous UBR5 induced MYC protein expression and regulated MYC target genes. Consistent with the tumor suppressor function of UBR5 (HYD) in Drosophila, HYD suppressed dMYC-dependent overgrowth of wing imaginal discs. In contrast, in cancer cells, UBR5 suppressed MYC-dependent priming to therapy-induced apoptosis. Of direct cancer relevance, MYC and UBR5 genes were coamplified in MYC-driven human cancers. Functionally, UBR5 suppressed MYC-mediated apoptosis in p53-mutant breast cancer cells with UBR5/MYC coamplification. Furthermore, single-cell immunofluorescence analysis demonstrated reciprocal expression of UBR5 and MYC in human basal-type breast cancer tissues. In summary, UBR5 is a novel MYC ubiquitin ligase and an endogenous rheostat for MYC activity. In MYC-amplified, and p53-mutant breast cancer cells, UBR5 has an important role in suppressing MYC-mediated apoptosis priming and in protection from drug-induced apoptosis. SIGNIFICANCE: These findings identify UBR5 as a novel MYC regulator, the inactivation of which could be very important for understanding of MYC dysregulation on cancer cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/7/1414/F1.large.jpg.