‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue for snRNAseq v3
Luciano G. Martelotto, Luciano G. Martelotto
Abstract
This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small sample sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al. This protocol is routinely used in the single-cell innovation lab for single nuclei experiments using 10x Genomics technologies. The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and tumors.