Resource Summary Report  New Search Previous Search Results

URL: https://dx.doi.org/DOI:10.17504/protocols.io.bq27myhn

Authors: Luciano Martelotto

Group: Human Cell Atlas Method Development Community, Single Cell Core, Harvard Medical School

Summary: 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 has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide 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 other samples types.Cardiomyocytes can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.

Affiliations: Harvard Medical School

External URL: https://research.unimelb.edu.au/centre-for-cancer-research/our-research/single-cell-innovation-lab

Version: 2

Publication Date: 2020

Expand All Collapse