| Popis: |
Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival. A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity. Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy. Here, we introduce scEC&T-seq (single cell extrachromosomal circular DNA and transcriptomic sequencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells. In this protocol, a single cell’s DNA is separated from its polyadenylated RNA as described by Macaulay et al. (2015)1. This is followed by removal of linear DNA through exonuclease digestion and further enrichment of circular DNA by rolling circle amplification with φ29 polymerase2-4. The separated mRNA from the same cell is processed using on-bead Smart-seq21. The duration of the entire procedure from cell sorting to library preparation is approximately 8 days. Our scEC&T-seq protocol has been validated in single cancer cells from neuroblastoma cell lines and primary tumors, and in normal single T-cells isolated from patient’s blood. Besides identifying large, oncogene-containing circular DNAs in cancer cells, our method also captures other smaller circular DNAs, which have been previously described in both cancer and non-malignant cells5. We envision that our method may enable the analysis of yet unknown prerequisites for the maintenance of both small and large circular DNA in cancers, but also in the context of other diseases and normal cellular development. |
